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THE APPLICATION OF A DIHYDROPYRIDINE-PYRIDINIUM SALT 
REDOX SYSTEM TO DRUG DELIVERY TO THE BRAIN 



MARCUS ELI BREWSTER III 



A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL 
OF THE UNIVERSITY OF FLORIDA IN 
PARTIAL FULFILLMENT OF THE REQUIREMENTS 
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY 



UNIVERSITY OF FLORIDA 



1982 



Copyright 1982 
by 

Marcus Eli Brewster III 



TO MOTHER 



ACKMOWLEDGEMENTS 

I would like to express my gratitude to Professor Nicholas S. Bodor. 
His genius and kindness will always be an inspiration to me. I would also 
like to thank the other members of my committee. Dr. Merle Battiste, 
Dr. Kenneth Sloan, Dr. Margaret James, and Dr. James Simpkins, for their 
advice and help. 

This work would truly not have been possible but for the guidance of 
a number of post-doctoral fellows and technicians, especially Dr. Hassan 
Farag, Dr. Cynthia Luiggi, Dr. Thorsteinn Loftsson, Mrs. Jirina Vlasak, 
Mrs. Nancy Gildersleeve, and Mr. Edward Phillips. I would also like to 
thank Jane and C.J. Rogers, Dr. Yasuo Oshiro and Dr. Tadao Sato for their 
assistance. 

Although space is limited, I want to recognize a few friends who 
made graduate school bearable: Linda and John Hirschy, Mark V. Davis, 
R.E. Golightly, Mrs. James E. Ray, Newton Galloway, Mike Morris, Richard 
Panarese, Wayne and Anita Riggins, Mike and Kay Dempsey, Chuck Harts field, 
Mike Gibbons, Raun and Cissy Kilgo, Jeff and Jane Dean, Ernie Lee, and 
the late Chip Connally. Chip was a dear friend. I would like to thank 
Scott and Margie Makar for their cordiality and hospitality. 

I would also to like to acknowledge the P.C.'s, Dr. Frank Davis, 
Mr. Jim Templeton, and Dr. Gary Visor, the last of a dying breed. Two 
special people deserve mention here because of their guidance early in my 
scientific career. Professor G.L. Ware, and Mrs. Frances Kenning. Special 
thanks are accorded to my Amazonian, sybaritic amanuensis, Cynthia Jordan. 
And, last but certainly not least, I would like to thank my family without 

iv 



whom I would not have been able to pursue an academic career. The antican- 
cer testing was graciously performed by Otsuka Pharmaceutical Co., Ltd. 
This work was supported by Grant GM 27167 from the National Institute of 
General Medical Sciences. 



V 



TABLE OF CONTENTS 

CHAPTER PAGE 

ACKNOWLEDGEMENTS iv 

LIST OF TABLES vii 

LIST OF FIGURES ix 

ABSTRACT xii 

1 INTRODUCTION 1 

Blood-Brain Barrier 2 

Prodrugs and Drug Delivery Systems 31 

Statement of the Problem 41 

2 MATERIALS AND METHODS 47 

Synthesis 49 

Characterization of Dihydroberberine 59 

Animal Studies 53 

3 RESULTS AND DISCUSSION 68 

Synthesis and Characterization of Dihydroberberine 68 

Theoretical Studies on the DihydropyridineJPyridinium 

Redox System 81 

Further Studies on the Chemical and Biological Properties 

of Dihydroberberine 104 

In Vivo Studies 116 

Conclusions 147 

BIBLIOGRAPHY 150 

BIOGRAPHICAL SKETCH 160 



Vi 



LIST OF TABLES 

TABLE PAGE 

1-1 Blood Brain Barrier Transport Systems 14 

3-1 Proton Assignments of the NMR of Dihydroberberine (2) 74 

3-2 Carbon Assignments of the NMR of Dihydroberberine (2) 76 

3-3 Distribution Coefficients for Berberine (1) and Dihydrober- 
berine Hydrochloride (3) in Chloroform/pH 7.4 Buffer and in 

1- Octanol/pH 7.4 Buffer 78 

3-4 The Heats of Formation, Vertical Ionization Potentials, and 

Dipole Moments of the Isoquinoline Model (30) and the Dihydro- 
isoquinoline Model (31) 86 

3-5 Bond Lengths in Angstroms between Various Atoms of the Iso- 
quinoline Model (30) and the Dihydroisoquinoline Model (31) 87 

3-6 Bond Angles in Degrees between Various Atoms of the Isoquino- 
line Model (30) and the Dihydroisoquinoline Model (31) 88 

3-7 Charge Density at Various Atoms of the Isoquinoline Model (30) 

and the Dihydroisoquinoline Model (31) 89 

3-8 Dihedral Angles between Various Atoms of the Isoquinoline Model 

(30) and the Dihydroisoquinoline Model (31) 90 

3-9 Differences in the Heats of Formations (AAHf) of (30) t (31), 

2- PAM t 1 ,4-Dihydro-2-PAM and 2-PAM Z 1 ,6-Dihydro-2-PAM 91 



3-10 A Comparison of Bond Lengths in Angstroms of the Pyridine {32) t 
1 ,2-Dihydropyridine (33) System and the Isoquinoline (30) ^ 
Dihydroisoquinoline (31) Model System 94 

3-11 A Comparison of the Bond Angles in Degrees of the Pyridine_^(32) ? 
1 ,2-Dihydropyridine (33) System and the Isoquinoline (30) ^ 
Dihydroisoquinoline (31) Model System 96 

3-12 A Comparison of the Atomic Charge Densities of the Pyridine {32) t 
1 ,2-Dihydropyridine (33) System and the Isoquinoline (30) t 
Dihydroisoquinoline (31) Model System 97 

3-13 The Rate of Oxidation of Dihydroberberine in Various Media 105 



vi i 



TABLE 



PAGE 



3-14 The Relative Rates of Oxidation of Dihydroberberine (2), 
1 -Methyl -1 ,4-dihydronicotinamide (21), and 1-Benzyl-l ,4- 
dihydronicotinaniide (22) in Dilute Hydrogen Peroxide 108 

3-15 Proton Assignments of the NMR of the 1-Methyl-l ,4-dihydro- 

nicotinic Acid Ester (27) "112 

3-16 The Rates of Oxidation and Corresponding Correlation Coef- 
ficients of Various 1-Methyl-l ,4-dihydronicotinic Acid Esters 
and 1-Benzyl-l ,4-dihydronicotinamide (22) 114 

3-17 The Effect of Glucose on the Movement of Berberine into Red 

Blood Cells lis 

3-18 Slow Infusion of Dihydroberberine (3) 131 

3-19 Efflux of 3H-Inulin from the Brain after Intracerebral Ventri- 
cular Administration 1-^^ 

3-20 In Vivo Metabolism of Berberine and Dihydroberberine in the 
RltT^PLC) 

3-21 In Vivo Metabolism of Berberine and Dihydroberberine in the 

RFtTTLC) I ^2 

3-22 Probit Analysis of the LD50 Study 145 

3-23 Effect of Berberine (1) and Dihydroberberine Hydrochloride (3) 

Against P388 Lymphocytic Leukemia 146 



vii i 



LIST OF FIGURES 



FIGURE PAGE 



1-1 This Schematic Illustration Represents an Endothelial Cell 
Derived from either a Muscle (EC^,) or Brain (EC,,) Capillary. 
In this Figure, (ma) is the Macula Adherens or Loose Junction, 
(zo) is the Zona Occludens or Tight Junction, (mv) are Micro- 
vesicles, and (bl) is the Basal Lamina. This Figure was Modi- 
fied from Reference 1, Page 162 by Permission 6 

1-2 A Proposed Carrier-mediated Chemical Delivery System with 
Specificity for the Brain. The Drug Molecule to be Trans- 
ported is Represented by the (O) 40 

1-3 The Proposed Drug Delivery Scheme 43 

3-1 Synthesis of Dihydroberberine (2) and its Hydrochloride 

Salt (3) 69 

3-2 Ultraviolet Spectrum of Dihydroberberine (2) in 95% Ethanol 70 

3-3 Infrared Spectrum of Dihydroberberine (2) (KBr) 71 

3-4 Mass Spectrum (70 eV, EI) of Dihydroberberine (2) 72 

3-5 Proton Nuclear Magnetic Resonance Spectrum (60 MHz) of Dihydro- 
berberine in CDCI3. The Insert Represents the Region between 
2.86 and 3.46 at 100 MHz 73 

3-6 The 13c Nuclear Magnetic Resonance Spectrum (100 MHz, CDCls) 

of Dihydroberberine (2) 75 

3-7 Demethylation of Berberine (1) and Methylation of Berber- 

rubin (4) 80 

3-8 Structures and Salient Numbering Protocols for Berberine (1), 
Dihydroberberine (2), the Isoquinoline Model (30), the Dihydro- 
isoquinoline Model (31), Pyridine (32), and 1,2-Dihydro- 
pyridine (33) 83 

3-9 The Highest Occupied Molecular Orbital of the Dihydroisoqui no- 
line Model (31) 92 

3-10 A Computer-assisted Drawing of the Most Stable Conformation 

of the Isoquinoline Model (30) at 25°C 99 



ix 



Figure Page 

3-11 A Computer-assisted Drawing of the Most Stable Conformation 

of the Isoquinoline Model (30) at 25°C. This View is Oriented 
so that the Interatomic Axis between Atoms 26 and 2 is Perpen- 
dicular to the Plane of the Page 100 

3-12 A Computer-assisted Drawing of the Most Stable Conformation 

of the 1,2-Dihydroisoquinoline Model (31) at 25°C 102 

3-13 A Computer-assisted Drawing of the Most Stable Conformation 

of the 1,2-Dihydroisoquinoline Model (31) at 25°C. This View 

is Oriented so that an Imaginary Axis between Atoms 2 and 5 

is Perpendicular to the Plane of the Page 103 

3-14 Spectral Changes of Dihydroberberine (2) upon Oxidation to 
Berberine (1) in pH 5.8 Phosphate Buffer at 26°C. Traces 
were made every 10 Min 106 

3-15 Proton Nuclear Magnetic Resonance Spectrum (60 MHz) of (27) 

in CDCI3 Ill 

3-16 The Rates of Oxidation of Various 1 -Methyl -1 ,4-dihydronicotinic 
Acid Esters (23). (24), (25), (26), (27), (28) and 1-Benzyl- 
1 ,4-Dihydronicotinamide (22) at 37°C in 40% Human Plasma (■), 
6% Brain Homogenate (A) and 3.5% Liver Homogenate (•) 113 

3-17 Partitioning of 26.5 mg of Berberine (1) from Plasma (A) into 
Red Blood Cells (A) and of 26.5 mg of Dihydroberberine Hydro- 
chloride (3) from Plasma (O) into Red Blood Cells (•). The 
Volume of Blood Used in each Experiment was 75 ml 117 

3-18 Distribution of Berberine in the Brain after iv Administration 
of Berberine (1) (♦) at a Dose of 55 mg/Kg or of Dihydrober- 
berine Free Base (2) (•) at a Dose of 55 mg/Kg 120 

3-19 Efflux of Berberine from the Brain after iv Administration of 
either 55 mg/Kg of Dihydroberberine Hydrochloride (3) (•) 
or 55 mg/Kg of Berberine (1) (♦) . Analysis was for (1) only 
and not Unoxidized (2) 121 

3-20 Efflux of Berberine (1) and Unoxidized Dihydroberberine (2) 
(A) after iv Administration of 55 mg/Kg of Dihydroberberine 
Hydrochloride (3) 122 

3-21 A Comparison of the Efflux of Berberine (1) (•) and Berberine 
(1) and Unoxidized Dihydroberberine (2) (A) after a Dose of 
55 mg/Kg of Dihydroberberine Hydrochloride (3) Administered 
iv 123 

3-22 Distribution of Berberine after iv Administration of 35 mg/Kg 
of Berberine (1) into the Kidney (♦) , Liver (■), Lung (•), 
and Brain (A) 125 



X 



Figure ^^9^ 

3-23 Distribution of Berberine after iv Administration of 55 

mg/Kg of Dihydroberberine Hydrochloride (3) into the Kidney 
(O). Liver (□), Lung (O), and Brain (A) 127 

3-24 A Comparison of the Efflux of Berberine from Lungs when Ad- 
ministered iv as 55 mg/Kg of Dihydroberberine Hydrochloride 
(3) (O) or 35 mg/Kg of Berberine (1) (•) 128 

3-25 A Comparison of the Efflux of Berberine from the Kidneys when 
Berberine (1) is Administered iv at a Dose of 35 mg/Kg (♦) 
and Dihydroberberine Hydrochloride (3) when Administered iv 
at a Dose of 55 mg/Kg «>) 129 

3-26 A Comparison of the Efflux of Berberine from the Liver when 
Berberine (1) is Administered iv at a Dose of 35 mg/Kg (fl) 
and Dihydroberberine Hydrochloride (3) when Administered iv 
a Dose of 55 mg/Kg (□) 130 

3-27 A Comparison of the Efflux of the Total Berberine, i.e. (1) 
and (2) from the Brain when either 55 mg/Kg of Dihydrober- 
berine Hydrochloride (3) is Administered iv (A) or 55 mg/Kg 
of Dihydroberberine (2) and 200 mg/Kg of 1-Methyl-l ,4- 
dihydronicotinamide (21) is Administered iv (•) 134 

3-28 Efflux of 1 -Benzyl nicotinamide Bromide (7) from the Brain af- 
ter iv Administration of 60 mg/Kg of 1 -Benzyl -1 ,4-dihydro- 
nicotinamide (22) (A) 135 

3-29 Efflux of Berberine from the Brain after icv Injection of 

either 50 yg of Berberine (1) (•) or 50 yg of Berberine (1) 
and 1000 yg of 1-Methylnicotinamide Iodide (6) (A) 137 

3-30 The LD50 Dose-response Curve of Berberine (1) (A) and Dihy- 
droberberine Hydrochloride (3) (•). Doses of (1) or (2) 
were Administered ip in CD-I Mice 144 



xi 



Abstract of Dissertation Presented to the Graduate Council 
of the University of Florida in Partial Fulfillment of the 
Requirements for the Degree of Doctor of Philosophy 

THE APPLICATION OF A DIHYDROPYRIDINE-PYRIDINIUM SALT 
REDOX SYSTEM TO DRUG DELIVERY TO THE BRAIN 

By 

MARCUS ELI BREWSTER III 
AUGUST 1982 

Chairman: Nicholas S. Bodor 

Major Department: Medicinal Chemistry 

This work has been concerned with the design and testing of a broadly 
applicable drug delivery system which is specific for the brain. The 
method developed for this purpose is based on a dihydropyridine:^yridinium 
salt redox system and on the blood-brain barrier. In the proposed delivery 
system, a pharmacologically active agent which contains a pyridinium nu- 
cleus would be reduced to its corresponding dihydropyridine. After sys- 
temic administration, the highly lipoidal dihydropyridine would partition 
into the brain as well as into the periphery. In both locations oxidation 
would occur. In the systemic circulation, the charge species would be 
rapidly eliminated by renal or biliary mechanisms while in the brain the 
compound would be retained. 

The prototype compound which was chosen for inclusion in this scheme 
was berberine. This alkaloid has a high in vitro activity against various 
cancer systems but its in vivo activity is low. The first step in the ap- 
plication of the described delivery system to berberine is the synthesis 



xii 



of dihydroberberine which was accomplished using sodium borohydride in 
pyridine. The dihydroberberine was analyzed by various spectroscopic means 
and a number of physical properties were measured. Theoretical calcula- 
tions using a MINDO/3 approach were also undertaken. 

In order to verify the proposed scheme, the delivery of berberine to 
the brain and the retention of berberine in the brain had to be shown. 
When dihydroberberine or its hydrochloride salt were injected systemi- 
cally, high levels of berberine were found in the brain and its efflux 
from the brain was slow. If, however, berberine is injected systemically , 
no detectable levels are found in the brain. When dihydroberberine is 
slowly infused the concentration of berberine rises in the brain and at 
forty- five minutes, this concentration is specifically higher in the brain 
than in any other organ analyzed. The mechanism of efflux of berberine 
from the brain was investigated and appears to be mediated by a passive 
process, perhaps the bulk flow of cerebral spinal fluid. Dihydroberberine 
was found to be less toxic than berberine. Preliminary anticancer data 
indicate that dihydroberberine is more effective in increasing the life 
span of animals injected intercerebrally with P388 lymphocytic leukemia 
than is berberine. 



xii i 



CHAPTER 1 
INTRODUCTION 

A method for delivering drugs specifically to a particular organ would 
be valuable. Properly designed, a drug delivery system should concentrate 
an agent at its site of action and reduce its concentration in other loca- 
tions. The results of these manipulations would not only be an increase in 
the efficacy of an agent, but also a decrease in its toxicity. 

A site specific system designed for the central nervous system (CNS) 
would be especially useful. The reason for this, aside from the inherent 
importance of the brain, is that the entry of many pharmacologically active 
agents into the CNS is impeded by a set of specialized barriers present at 
the blood-brain interface. This barrier system, termed the blood-brain 
barrier (BBB), is composed of numerous enzymatic and anatomical components. 

This dissertation will present a general method for the specific deliv- 
ery of drugs to the brain and give an example which substantiates the method. 
In order to provide an adequate background for a discussion of drug deliv- 
ery to the brain, a review of the BBB is necessary. The introductory mate- 
rial is then continued with a cursory historical account of drug delivery 
systems and prodrugs. Because of the importance and great interest of an- 
ticancer agents, emphasis is placed on this topic in this section. The 
closing section of the first chapter will state specific aspirations as they 
apply to the present research. 



1 



Blood-Brain Barrier 
Structural and Enzymatic Considerations of the Blood-Brain Barrier 

The existence of a barrier which separates central nervous tissue 
from the general circulation was first postulated by Ehrlich at the end 
of the 19th century. ^'^'^''^'S g series of pioneering experiments, he 
injected a number of dyes into laboratory animals and found that, while the 
visceral organs were highly stained, the brain was conspicuously uncolored. 
It was later discovered that these dyes bind extensively to plasma proteins 
so that the actual barrier then described was one to these complexes. Many 
small hydrophilic compounds cannot, however, pass into the brain so that this 
barrier is presented to a wide variety of compounds. Early in the study of 
the BBS, the obstruction was considered absolute but this idea was soon dis- 
pensed with since the nutritional requirements of the brain necessitate 
the equilibration of a number of compounds between the general circulation 
and the CNS.3 

The morphological basis of the BBB had been a very controversial sub- 
ject until relatively recently. Historically, three hypotheses have been 
put forward to explain this impermeability to blood-borne substances. ^ 
All are based on structural differences between the cerebral vascular sys- 
tem and the systemic circulation. It was proposed that the small extra- 
cellular space characteristic of mammalian brains prohibited the accumula- 
tion of compounds and, as such, constituted a barrier. It was shown, how- 
ever, that some animals with large extracellular spaces have a wel 1-defined 
barrier to a number of substances.^ The suggestion was also made that the 
general impermeability of the brain to blood-borne substances was due to 
astrocytic end feet which surround the capillaries, forming an envelope, 
or due to the endothelial cell lining of the cerebral capillaries.^ In 
order to study this question, electron microscopic evaluation is necessary. 



Progress was somewhat slowed because of the lack of appropriate electron 
microscopic tracers. ^ The first tracers used were saccharated iron oxides^ 
or ferritin (molecular weight 560,000) whose limits of resolution were close 
to the thickness of the endothelium itself. It was not until the intro- 
duction of horseradish peroxidase (HRP) and microperoxidase (MP) that the 
exact structure of the BBB could be deduced. Horseradish peroxidase is a 
relatively small enzyme (molecular weight 43,000) which, unlike ferritin, 
does not contain an electron-dense core but rather produces a material 
which has a high affinity for osmium tetraoxide and other radiopaque sub- 
stances.'' 

By using HRP, Reese and Karnovsky demonstrated the inability of the 
marker to pass from the lumen of the cerebral capillary.^.io fact, HRP 
was never found in the extracellular space surrounding the capillary. Addi- 
tionally, when HRP was injected directly into the brain it readily passed 
the astrocytic end processes and was stopped at the endothelial membrane. 
The anatomical basis of the BBB was, therefore, isolated to the endothelial 
lining of the cerebral capillaries and not a perivascular site. There are 
several ultras tructural differences between systemic capillaries and cere- 
bral capillaries which account for their general impermeability. ^'"^ 

The manner in which endothelial cells of the cerebral capillaries are 
joined is distinct from systemic capillaries. Cerebral junctions are char- 
acterized as tight or closed junctions meaning the cells closely approximate 
each other. These junctions gird the cell circumferentially, forming a 
zona occludens and providing an absolute barrier to HRP. Structurally, 
the junctions consist of aligned intramembranous ridges and grooves which 
are in close apposition. These tight junctions have been examined by thin 
section electron microscopy and attempts are now underway to examine them 
by a freeze- fracture technique.'^'^^ jhis method, which allows a longitudinal 



view of the capillaries, will add greatly to the structural knowledge of 
these tight junctions but the method is technically difficult. Recently, 
a freeze-fracture technique was applied to the cerebral vasculature of a 
chameleon, which possesses a BBB similar to that of mammals. in these 
animals a series of ridges and grooves is seen. The ridges are connected 
to neighboring ridges by an anastomosing network. In general, the more com- 
plex this system is, i.e. the number of ridges it has, the tighter the junc- 
tion is. The tightness of the junction can be assayed not only structurally 
but also by measuring ionic conductance and resistance through the junction. 
The ionic conductance is low for most ions.^^'^'^ 

Systemic capillaries lack this closed junction. Morphologically, this 
can be traced to a lack of continuity in the intercellular appositions. 
In cardiac muscle, for example, the ratio of junctional width to the width 
of the cell membrane is 2.4, while in cerebral capillaries this is reduced 
to 1.7.1° jhese open junctions allow a high degree of nonspecific transport 
of nutrients and other compounds into systemic capillaries. Materials pass 
easily between these leaky cells, while in the brain the sealing of the 
intercellular fissures severely restricts this nonspecific transport. Since 
intercellular transport is removed, only intracellular transport remains. 
Lipophilic compounds can readily pass through these phospholipoidal membranes, 
but hydrophilic compounds and compounds with high molecular weights are ex- 
cluded. Systems are available to transport small hydrophilic nutrients, 
and this will be discussed later. 

A second difference between cerebral and systemic capillaries is the 
paucity of vesicles and vesicular transport in the CNS.'^'^ Vesicular trans- 
port is a process for transcellular transport and, as such, vesicles are 
transported from the luminal to the abluminal membrane. Pinocytotic acti- 
vity, on the other hand, is concerned with the nutritional requirements of 



5 



the cell and, therefore, involves vesicular movement from the luminal mem- 
brane to a cell organelle, presumably a lysosome. 

Cerebral endothelial vesicles are usually uncoated and few in number 
compared to other systems. Using electron microscopic morphometry, five 
vesicles per micrometer luminally and thirty- forty vesicles per micrometer 
abluminally were found.'* In the diaphragm and in myocardial vessels the 
values are much higher, being seventy-eight and eighty-nine vesicles per 
micrometer, respectively. This lower content of vesicles is another mecha- 
nism by which the CNS can limit nonspecific influx. A third difference 
is the lack of fenestra in the cerebral capillaries. These differences 
are demonstrated schematically in Figure 1-1. 

Cerebral vessels have a number of perivascular accessory structures 
which appear to be involved in BBB function.*^ While it is known that the 
astrocytic end feet are not involved as a barrier per se, their role in 
attenuating BBB action is interesting. These glial end feet may be involved 
with regulation of amino acid flux. They also appear to engulf protein 
which breeches the BBB and, therefore, may act as a second line of defense.^' 
Phagocytic pericytes which are present abluminally may play a similar role. 
It is possible that the basement member of endothelial capillaries acts as 
a mass filter preventing large molecules from penetrating it. 

In addition to these structural features the BBB maintains a number of 
enzymes which appear to augment barrier function. 2. is, 17 since optimal 
neuronal control requires a careful balancing between neurotransmitter 
release, metabolism, and uptake, it is of vital importance to restrict the 
entry of blood-borne neurotransmitters into the CNS. It is not surprising, 
therefore, to find high concentrations of such enzymes as catechol -0-methyl 
transferase (COMT), monoamine oxidase (MAO), Y-aminobutyric acid trans- 
aminase (6ABA-T) and aromatic amino acid decarboxylase (DOPA decarboxylase) 



6 



"I 




1-1. This Schematic Illustration Represents an Endothelial Cell 
Derived from either a Muscle (ECm) or Brain (ECb) Capillary. 
In this Figure, (ma) is the Macula Adherens or Loose Junction, 
(zo) is the Zona Occludens or Tight Junction, (mv) are Micro- 
vesicles, and (bl) is the Basal Lamina. This Figure was Modi- 
fied from Reference 1, Page 162 by Permission. 



7 



in the BBB. Recently, a distributional study of COMT in the brain indi- 
cated that this enzyme is present in many sites including several which 
lack a structural BBB.i^ This distribution may aid in the exclusion of 
neurotransmitters from structurally unprotected areas. The presence of 
DOPA decarboxylase explains partially the need for giving such large doses 
of L-dihydroxyphenyl alanine (DOPA) in the treatment of CNS dopamine defi- 
ciencies to achieve appropriate therapeutic cerebral levels. The enzymatic 
BBB may also play a role in the exclusion of lipophilic compounds which 
might otherwise passively diffuse through the BBB. This is suggested by 
the presence of pseudo (butyryl) cholinesterase in the cerebral capil- 
laries.** This enzyme is not found in nonce rebral capillaries. The occur- 
rence of Y-glutamyl transpeptidase has also been described and may account 
for some protection from peptide infil tration.^ »19 An early proposal 
that Y-glutamyl transpeptidase is involved with carrier systems is ques- 
tionable. Acid phosphatase activity, which is a marker for lysosomes and 
pre-lysosomes or phagosomes, is present in the endothelial cells.^^ These 
organelles appear to be involved in the degradation of endocytosed material 
and, as such, can be considered a component of the BBB. The endothelial 
cells of the cerebral microcirculation contain a large number of enzymes 
but care must be taken in ascribing a certain enzyme to a barrier role.^'^ 
There are numerous enzymes which, while present in the endothelial cells, 
do not serve in any capacity other than general cellular functioning. 
Molecular Carriers Involved in BBB Transport 

The aspects of the BBB which have been discussed thus far give an indi- 
cation of its relative impermeability to a number of blood-borne substances, 
but do not explain the movement of essential nutrients into the CNS. This 
transport is brought about by a number of carriers which are situated in 
the endothelial cells. These carriers are generally assumed to be proteinaceous. 



8 



They are equilibrati ve, i.e. nonenergy dependent and bidirectional in 
nature and can be saturated. ^ '2° '^i The net movement of compounds is 
always along a concentration gradient and since nutrients are readily 
utilized as soon as they pass into the brain, this gradient is in the di- 
rection of the brain. 

A number of specific carriers for compounds have been described. The 
first to be characterized was one for hexoses . ^ '^^ ,22 j^-js carrier displays 
saturable kinetics and can be competitively inhibited. The hexose carrier 
is stereospecific and has a high affinity for a-D-glucose with a Km between 
6.0 and 9.0 mM. Other sugars with affinity for this carrier include, in 
order of decreasing affinity, 2-deoxy-D-glucose, 3-0-methyl glucose, B-D- 
glucose, D-mannose, D-galactose and D- xylose. 22 The Km of D- fructose and 
L-glucose is very high. 

The carrier is Na"*" independent and is inhibited by phloretin, a non- 
competitive inhibitor, more than phlorizin, a competitive inhibitor. 2° 
The V,^ax ''S similar for all sugars tested which indicates the rate limiting 
step for transport is not the association of the sugar with the carrier but, 
rather, the movement of the carrier across the membrane complex. This car- 
rier demonstrates exchange diffusion, i.e. the carrier moves more rapidly 

2.2. 2.3 

when loaded than when empty. 

At a Km of 7 mM, the concentration of glucose required to produce sat- 
uration is about 126 mg% so that under physiological conditions, the system 
is about half saturated. In normal situations the rate determining step in 
glucose utilization is the hexokinase step. This can be shifted to BBB 
transport of glucose in conditions of hypoglycemia. 21 While substrate flux 
is usually thought of as being related only to plasma levels of glucose, 
recent studies have indicated that intracerebral glucose concentrations can 
alter carrier kinetics. 



The effects of insulin on the transport of glucose are controversial 22> 
Several reports have indicated no effect on either unidirectional or net 
flux after insulin infusion. This is curious in that insulin would not be 
expected to pass the BBB. The presence of insulin receptors in cerebral 
capillaries may explain this enigma in that insulin may bind to a receptor 
on the luminal surface and its effects may be mediated to the abluminal 
surface by a second messenger. 2'+ 

Also controversial is the possibility that a low and high affinity 
system is operating in glucose transport. 2° There is a nonspecific 
flux associated with glucose of 1%. Some authors attribute this to diffu- 
sion but an alternate hypothesis has been proposed. This involves the pres- 
ence of two systems on the carrier: a high affinity, low capacity system 
and a low affinity, high capacity system. Most data have been collected in 
isolated vessel preparation but there is some support of this proposal from 
in vivo experiments. Gjedde showed the putative low affinity system to be 
stereoselective and to have a Km of 1.0 M,23 compared with a Km of 1.1 mM 
for the high affinity system. He contends that a single set of kinetic 
parameters does not adequately describe the system and that this high-low 
affinity system better correlates vyith the data. A suggestion that a pro- 
tein tetrameter with both low and high affinity sites was the carrier has 
also been made. At the choriod plexus there appears to be ouiban sensi- 
tive, Na"*" dependent glucose flux and this is apparently important in cere- 
bral spinal fluid (CSF) homeostasi s . 22 

Three carriers have been described for amino acid transport. ^ '^2.20 
These carriers have affinity for neutral, basic, and acidic amino acids. 
In general, essential amino acids, which are large and bulky, are trans- 
ported in preference to nonessential amino acids. in all of these 



' 10 

systems, net flux is small compared to unidirectional flux since amino 
acids derived from proteins are constantly being lost and the magnitude 
of this loss is similar to uptake. 

The transport of neutral amino acids has been described by Christensen 
and these generalizations apply to the BBB.25,26 pour neutral amino acid 
transport systems have been shown to occur in Ehrlich ascites, a model cell 
system, and in several other systems. An L- or leucine-preferring system 
is characterized by high affinity for phenylalanine, leucine, tyrosine, 
tryptophan and several other large essential amino acids. It is Na"*" inde- 
pendent, bidirectional and equilibrative in nature. The definition of this 
system can be made by observing the flux of 2-aminonorbornane-2-carboxylic 
acid which is exclusively transported by this carrier. The A- or alanine- 
preferring system is characterized by a Na"*" dependence, an energy depen- 
dence, and the ability to concentrate substrates. The system has affinity 
for glycine, proline, alanine, serine, threonine and several other small 
amino acids. The defining compound for this system is a- (methyl ami no) - 
isobutyric acid. Two other amino acid transport carriers have also been 
described but they are not well characterized. There is also an ASC 
(alanine-serine-cysteine-preferring) system and a Gly (glycine-preferring) 
system. 

Since generally only large essential amino acids (L-system substrates) 
are transported through the BBB, the L-system is assumed to be the dominant 
mechanism in amino acid uptake. i> 2° The absence, however, of the A-system 
has been questioned. It was recently argued that the A system is present 
but has a different distribution than the L-system. Betz and Goldstein dis- 
covered a system whose characteristics are similar to the A system but which is 
located abluminally.^^ This system may act as an active mechanism for 



efflux of these amino acids from the brain parenchyma or, more probably, 
for concentrating them in the endothelial cytosol . The necessity for 
this concentration is related to a proposal that the L and A systems may 
act together in the transport of amino acids. This hypothesis is based on 
a possible equilibration of amino acids between the two carriers in the 
cytoplasm of the endothelial cell.^^ 

In many cases the Km value for an amino acid is similar to its plasma 
concentrations. This being the case, slight changes in the blood levels 
of an amino acid may alter their disposition. The utilization of trypto- 
phan, for example, which is a precursor of serotonin, is partially deter- 
mined by its availability and its movement across the BBB.2i'28 a similar 
situation may exist in certain circumstances with tyrosine, which is the 
precursor for dopamine, norepinephrine and epinephrine. 

Recently, a hypothesis was forwarded to explain the regulation and 
induction of these carriers. 25»30, 3 i n been suggested that an amino 
acid is produced abluminally in large amounts and transported on the neu- 
tral amino acid carrier. The amino acids in such a system should have a 
high Km and, therefore, be easily displaced from the carrier at the luminal 
side or in transit. These carriers, like the hexose carriers, exhibit ex- 
change diffusion. The proposed amino acid in this regulatory role is 
glutamine. Glutamine is synthesized from glutamic acid and ammonia by 
glutamine synthetase in astrocytes, a perivascular locus. The concentra- 
tion of glutamine is ten times higher than the concentration of any other 
neutral amino acid and its local concentration in the vicinity of the BBS 
is predicted to be still higher. 

This hypothesis was formulated on the basis of several interesting 
observations. In cases where ammonia and, presumably, glutamine levels 



12 

increase cerebral ly, as in porto-systemic shunts, the uptake of phenyl- 
alanine and other neutral amino acids increases. Also, if glutamine syn- 
thetase is inhibited by methionine sulfoximine, the uptake of essential 
amino acids is reduced. ^9 While this is an attractive proposal, several 
authors have questioned it on the grounds that many amino acids compete 
for this neutral amino acid carrier and the role of glutamine, therefore, 
may be relatively unimportant. ^ 2 

Relatively little work has been done with basic and acidic amino 
acids. The acidic system has affinity for glutamate and is fully saturated 
at physiological concentrations. Basic amino acids are transported on a 
distinct carrier. This carrier is equil ibrati ve, saturable and bidirec- 
tional and has been described as a Ly"^ or lysine-preferring system. 

A monocarboxylic acid carrier has been described which demonstrates 
affinity for lactate, pyruvate, acetate, propionate, butyrate, 5-aminolevu- 
linic acid and ketone bodies. ^'^o Ketone bodies, such as 6-hydroxybutyrate 
and acetoacetate, are produced in a number of stressful circumstances, 
including starvation. ^3 The carrier possesses similar characteristics to 
those which have already been described. It is stereospecific and pH sen- 
sitive. 20 The transport of the substrates on the carrier increases with a 
decrease in pH. This has been interpreted to mean either that a proton is 
cotransported with the acid or that hydroxide acts as a high affinity com- 
petitive inhibitor. Only the ionized acid is transported by this system 
and this has led to the proposal that an R-NHg"*" moiety is present in the 
active site. The unionized acid can pass through the membrane by simple 
diffusion. 

The Km of lactate is similar to the plasma level of lactate. This 
being the case, rapid rises in systemic lactate levels, such as the ones 
which accompany exercise, are not transferred to the brain immediately. 



13 

A carrier for choline was described in 1978. Choline cannot be 
synthesized de novo in the brain but this precursor is required for such 
important cellular components as acetylcholine and phosphatidylcholine. 
The carrier conforms generally to those characteristics specified for other 
systems. It has affinity for choline, hemicholinium, dimethyl ami noethanol 
(deanol), tetraethyl ammonium, tetramethyl ammonium, cartitine and spermine 
but not for NH^"^. The distribution of this carrier is not uniform. Choline 
uptake decreases with age and this correlates with an age-dependent diminu- 
tion of the carrier. The rate determining step in choline utilization is 
its movement across the BBB. 

A carrier with affinity for nucleosides such as adenosine, guanosine 
and inosine has been described. ^ '2° Additionally, a system for transport- 
ing purine bases has been isolated. This system transports adenine, guanine 
and hypoxanthine but not pyrimi dines. This is interesting in light of the 
fact that pyrimi dines can be synthesized in the brain from NH^"*" and aspar- 
tate, while purines cannot. This carrier is very active in neonates, but 
its activity diminishes with age.^^ 

Recently, a carrier was described for thyroid hormones. 36> 37 jhg 
carrier has affinity for triiodotyrosine (T3) and thyroxine (T^) but not 
for tyrosine, leucine or potassium iodide. The transport of T3 is satur- 
able and inhibited by T^. The carrier is weakly stereospecific and of high 
affinity. The presence of a carrier for T3 and T^ is interesting because 
these compounds are fairly lipophilic. The bulkiness of the molecule, 
however, tends to decrease its ability to pass membranes. A carrier- 
mediated transport has also been proposed for thiamine. These systems 
are summarized in Table 1-1. 



14 



Table 1-1 . Blood-Brain Barrier Transport Systems 



Representative Km "^max 

Transport system Substrate (mM) (nmol min'^g'^) 



Hexose 


61 ucose 


9 


1600 


Neutral Amino Acid 


Phenylalanine 


0.12 


30 


Acidic Amino Acid 


Gl utamate 






Basic Amino Acid 


Lysine 


0.10 


6 


Monocarboxyl ic Acid 


Lactate 


1.9 


120 


Amine 


Choi ine 


0.22 


6 


Nucleoside 


Adenosine 


0.018 


0.7 


Purine 


Adenine 


0.027 


1 


Thyroid Hormone 




0.001 


0.17 


Thiamine 


Thiamine 







15 

In addition to these carrier systems, there are a number of active 
efflux mechanisms. Two of these systems are located in the choriod plexus 
and have affinity for organic ions. A system for the disposition of anions 
has been described and divided into two subsystems.20»35''*0''*i''+2 An L 
or liver system with affinity for prostaglandins, 5-hydroxyindole acetic 
acid and probenicid, as well as a K or kidney system with affinity for 
p-aminohippuric acid and phenol red, has been proposed. Additionally, a 
carrier for the efflux of iodide has been described.'*^ A cationic sys- 
tem is also present in the choriod plexus and this species has affinity 
for N-methyl nicotinamide, decamethonium and hexamethonium ions.'*5»'+5 
These systems appear to be important in the removal of metabolic acids and 
bases. This, as well as the anionic system, is energy-dependent and can 
be competitively inhibited. The presence of several energy (ATP) -dependent 
systems in the capillaries indicates that this site may also be important 
for active efflux. The high density of mitochondria in cerebral capil- 
laries is further support for this location.'*^ 
Movement of Compounds Across the BBB 

The BBB, therefore, consists of a relatively impermeable membrane 
superimposed on which are mechanisms for allowing the entrance of essential 
nutrients and the exit of metabolic wastes. If a compound is to gain access 
to brain parenchyma, it may do so via several routes. If the agent has 
affinity for one of the carriers previously described, it may diffuse across 
the BBB by association with this carrier. A compound which has a high in- 
trinsic lipophilicity can diffuse passively through the phospholipoidal 
cell membrane matrix.^''^'^^ jhg pK of a compound with ionizable groups is 
also important, since only the unionized species diffuses across the BBB 
rapidly. The ability of a substance to enter into the cell membrane 



16 

is often correlated with its in vitro octanol :water partition coefficient.50»^ 
This is a measure of 1 ipophil icity, and can be correlated with biological 
effects. 52 jhese correlations can be extended to the permeability of com- 
pounds through the BBB. Several examples of this correlation have appeared 
in the literature, including the opiates morphine, codeine, and heroin. 
Good correlation is obtained between 1 ipophil icity, the ability to pass 
the BBB, and narcotic efficacy. 

The increase in the ability of a compound to pass membranes can, all 
too often, be correlated with an increase in undesirable side effects. An 
example of this was shown in a series of B-blockers in which lipophilic 
members of this pharmacologic group, i.e. propranolol penetrated the BBB 
rapidly but demonstrated a number of deleterious psychiatric manifestations.^' 
Conversely, B-blockers of lower lipophilicity exhibit an attenuation of 
these side effects. 

These two avenues, namely passive diffusion and carrier mediation, 
represent the major components of influx. Other minor mechanisms may 
also allow the entry of substrates into the CNS. The cell bodies of many 
neurons are located centrally while their axons may penetrate into the 
periphery. These axons can take up material and transport it in a retro- 
grade fashion to the CNS.^'S'* This retrograde axoplasmic transport has 
been observed in such areas as the nucleus ambiguus and the abducen nucleus. 
In some cases, however, the endocytosed material is reacted with lysosomes 
and, therefore, this transport route may have a protective function. 

There are several areas of the brain which lack a BBB.^'^'^^ These 
include such locations near the ventricles as the area postrema, the sub- 
fornical organ, the median eminence of the neurohyphosis , the organum vas- 
culosum of the lamina terminalis, and the choriod plexus. Collectively, 



17 

these areas are termed the circumventricul ar organ. In addition, the 
pineal gland lacks a BBB. These areas constitute a small fraction of the 
total surface area of the BBB and may allow a limited nonspecific flux. 

These loci do have important pharmacological ramifications as demon- 
strated by the action of a series of atypical neuroleptics.^e.sy j^gse 
compounds are so named because they provoke certain symptoms of dopaminer- 
gic blockade but not others. Specifically, metoclopramide exerts an anti- 
emetic action but not an anti schizophrenic effect. This dichotomy was 
explained by the fact that metoclopramide does not penetrate the BBB. The 
site of action of antiemetic agents is at the chemo receptive trigger zone, 
which is located in the area postrema and is outside the BBB. The apparent 
inability of metoclopramide to produce the full spectrum of changes which 
occurs as a consequence of dopaminergic blockade may be related to its 
pharmacokinetics and, specifically, its inability to pass the BBB. This 
relegates the compound to only those sites of action not protected by the 
BBB. Other pharmacologically important sites outside the BBB include the 
median eminence, which controls prolactin secretion. In some areas, while 
the BBB is not absent it is diminished. These areas include certain ar- 
teriolar segments whose diameters are between 15-30 ym. ' ' In these 
segments limited protein extravasation, or leakage of compounds from the 
lumen of the capillary to the extracellular spaces, has been observed. 

The ability of small peptides to penetrate the BBB is an extremely 
controversial point. '^^ '^^ Systemic administration of certain cen- 
trally active peptides elicits a central response. Differences concerning 
the interpretation of these data are significant. One view is that the flux 
of these small peptides across the BBB is low indicating, perhaps, some 
nonspecific route accounts for their entry. 21 '62 j^g presence of peptidyl 



18 

receptors luminally, whose stimulation results in the generation of a 
second messenger, has been proposed in this respect. A different conclu- 
sion is that small peptides have a significant flux across the BBB, The 
peptides which have been investigated thus far include stabilized enkepha- 
lins and endorphins, a nonapeptide which induces delta-sleep, melanin stim- 
ulating hormone (a-MSH) and melanin inhibiting factor one (MIF-1 ) .6° 

The BBB plays a major role in CSF homeostasis. . 6k Cerebral spinal 
fluid is produced at the choriod plexus and drains from the ventricals 
through the foramina of Magendie and Luschka into the ventral aspects of 
the brain. 65 jh-js fluid serves many important mechanical and nutritive 
functions. Cerebral spinal fluid flow is constant with a ti of renewal 

2 

of about two hundred and seventy minutes. The ventricular volume of CSF 
is about 23 ml while the subarachnoid volume is 117 ml. Cerebral spinal 
fluid, along with any dissolved materials, leaves the subarachnoid space 
via the arachnoid villi, which protrude into a venous sinous. The arach- 
noid villi act as a one-way valve and prevent backflow.^^ This loss of 
CSF provides a slow mechanism for nonspecific efflux of compounds from 
the CNS. This mechanism rids the brain of polar compounds such as metabolic 
wastes at a fairly constant rate regardless of molecular weight. If a 
compound is fairly polar and does not have affinity for any passive or 
active efflux mechanism, it will leave the CNS by CSF bulk flow. Therefore, 
while lipophilicity is very important for influx to the brain, the efflux 
of a compound is only partially dependent on this parameter. '6''' '6Q'6^»''° 
Since the ionic environment in which neurons function is so important, 
the composition of the CSF is strictly maintained within narrow limits. 
The CSF is not simply an ultrafi Itrate of plasma, and several concentration 
gradients are produced. The maintenance of these gradients can, in part, 
be attributed to the low ionic conductance of the BBB. This is especially 



19 

important for K+, since a low K^sf/K+p^gs^^ aPPa^^^^ly ^^^^ *° 

stabilize neurons. ''^ ''^^ 

One of the major factors in influencing diffusion into the CNS is the 
degree to which a substance is bound to plasma proteins. It had been 
assumed for a long time that only the free, dialyzable fraction of the 
total plasma concentration of a compound was available for diffusion. ^ 
This is important for a great number of compounds. The major species in- 
volved in this binding are serum albumins, which tend to bind molecules 
loosely but to a large extent, and globulins, which bind with high affinity 
and low capacity. The premise that only the unbound species is capable 
of diffusion has, however, been challenged. 

Steroids have profound central effects and gain entry into the CNS 
by simple diffusion across the BBB, even though they are highly bound to 
plasma proteins. ^ This diffusion correlates well with the octanol :water 
partition coefficient of the steroids and inversely with the tendency of 
the molecules to form hydrogen bonds. Different steroids are taken up 
differently, however, and this is in large part related to protein binding. 
There are several globulins which bind specific steroids. These include 
sex hormone binding globulin (SHBG), which is found in man but not rats, 
cortical binding globulin (CBG), estradiol binding globulin (EBG) which, 
in fetal and neonatal rats, may be synonymous with a-fetoprotein, proges- 
terone binding globulin (PBG), which is found in pregnant guinea pigs and 
also, thyroid hormone binding globulin (TBG), which is found in man. 

Those steroids which are bound to globulins such as cortisone to CBG 
in rats have a small flux into the brain while those steroids which are 
bound more highly to albumins such as progesterone, estrogen or testosterone 
in the rat, easily diffuse through the BBB.^^ jhe reason for this is that 



20 

binding to albumin is sufficiently weak that the capillary transit time 

in the brain is long enough to allow dissociation of the compound from 

the macromolecule. In the case of globulins, however, the binding is 

tighter and the turnover is only of the order of 3-10/o/second. The sojourn 

through the cerebral capillaries is not, therefore, sufficient to release 

the bound material. It is not, therefore, the plasma-protein-bound frac- \ 

tion which is unavailable for transport but, rather, the globulin-bound i 

fraction. 

These principles also apply to free fatty acids such as palmitate. 
In this case, however, there appear to be high and low affinity sites on 
the albumin molecule so that the rate of dissociation of the palmitate 
bound to the site with the lower Km may be slow compared with capillary 
time, while the low affinity site may not.'^'* Melatonin, which is also 
highly protein bound, shows a significant diffusion through the BBB.^^ 

Thyroid hormones which are transported into the CNS by carriers are 
also bound by plasma proteins. ^^jS^ The carrier, whose Km is lower than 
that of the albumin site, is able to compete successfully with the albumin 
for binding. This is effectively a stripping of the compound from its 
albumin site. The effect is also seen with tryptophan and other amino 
acids. 28 

The BBB in Pathological and Experimentally Altered States 

The integrity of the BBB is known to be impaired in a number of patho- 
logical or experimentally-induced conditions.'*'^^ '^'^ The effect produced 
can be the result of changes of the structural components of cerebral capil- 
laries such as the junction or vesicular activity and, as such, results 

in generalized increases in permeability. Alternatively, the carrier sys- .\ 

•I 

terns may be compromised and this may lead to specific changes in perme- ; 
ability. 



21 

Generalized increases in permeability result in a number of deleteri- 
ous events. Since the BBB is relatively permeable to water, but not to 
most other substances, osmotic gradients can be rapidly changed. If plas- 
ma proteins and other compounds are allowed to freely enter the CNS, they 
will bring with them large amounts of water, with the result being cere- 
bral edema. ^'2° 

The morphological basis of these changes in any particular situation 
can be highly controversial. This is especially the case with hypertonic 
treatment of cerebral capillaries. It has been known for some time that 
hypertonic solutions of such solutes as glucose, sucrose, urea, arabinose, 
lactamide and several others can increase the permeability of the cerebral 
capillaries to protein and other small polar compounds. Rapoport explains 
this phenomenon as the result of osmotic shrinkage of the endothelial cells, 
resulting in a pull ing apart of the tight junctions. ^'^^ This has been 
challenged. If HRP is injected after a hypertonic solution, the HRP reac- 
tion product does not form a continuous line from the luminal to the ablu- 
minal surfaces at the junction or at any other location.'* '^o n was sug- 
gested, therefore, that increased vesicular transport accounted for the 
increased permeability, perhaps as a result of increases in local blood 
pressure.'^'' 

In any case, if the concentration of the hypertonic solution is close 
to the critical opening concentration, the opening of the BBB is transient 
and does not produce acute edema. ^ This procedure may have therapeutic 
applications. In many cases it is desirable to introduce highly polar 
compounds into the brain and this osmotic opening of the BBB may provide 
an avenue for that purpose. Methotrexate is a folate inhibitor used in the 
treatment of cancer. The pKg of the carboxylic acid functions of this 



22 

molecule is 4.7 and at physiological pH methotrexate is 99.8% ionized and, 
as such, passes the BBB very slowly, if at all. After pretreatment with 
hypertonic arabinose, the concentration of methotrexate showed a fifty- fold 
increase in the CNS.^^ 

Hypertension is a major health problem and can produce a number of 
debilitating complications.^^ Hypertension increases the extravasation 
of albumin, sucrose, and other polar compounds. 3° '^^ '^^ jhis has been 
studied in a number of animal models of hypertension including those in 
which the increased system blood pressure is induced with either ampheta- 
mine, ephedrine, Aramine or bicuculline. 

The basis for this increased permeability has been debated frequently 
and appears to be related to increased vesicular transport since ultra- 
structural investigations do not indicate capillary lesions or junctional 
openings. ''■^ Increased vesicular transport has, in fact, been implicated 
in a number of situations in which capillary permeability increases. The 
factors that affect vesicular formation which are described here also ap- 
ply in those cases. 

The mechanism by which hypertension elicits an increase in vesicular 
activity is not clear. The increased hydrostatic pressure may act to 
induce an invagination. Also, there are a number of substances associated 
with hypertension that induce vesicular formation. '♦'^^'^^ jhese include 
the catecholamines, serotonin, and histamine. Joo explained the increased 
vesicular transport in terms of a cyclic 5'-adenine monophosphate (cAMP) 
stimulation mediated by a catalyzed adenyl cyclase, since cAMP can directly 
induce vesicular formation.^'*'^^ 

Both serotonin and histamine have been shown to increase protein ex- 
travasation and both act to catalyze specific adenyl cyclase. A perivascular 



23 

source of both of these compounds is available, since histamine is stored 
in perivascular mast cells and serotonin in platelets.'* The action of 
histamine is partially reversible by H2 antagonists. Recently, cyclic 
guanine monophosphate (cGMP) has been isolated in cerebral capillaries and 
this may play a role in vesicular transport. These effects are seen 
peripherally as well as centrally. 

The major controversy surrounding this area is whether hypertension 
itself is responsible for extravasation, or if some humoral agent produced 
as a result of hypertension is culpable. It is difficult to look at this 
in vivo since many agents can increase blood pressure and increase vesi- 
cular transport. 

Additional evidence that vesicles are important in extravasation is 
indicated by the decreased protein flux in capillaries treated with com- 
pounds which decrease vesicular formation. These compounds include imida- 
zole, which alters cAMP function by inhibiting the inactivation of phos- 
phodiesterase, thioridazine, a phenothiazine which decreases vesicular 
fusion with the cell membrane, and desipramine.^° The anionic transport 
blocking agent 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid 
disodium (SITS) inhibits exocytosis . This compound, which also inhibits 
protein extravasation in hypertension, inhibits ATP-evoked adrenalin 
release from chromaffin granules as well as serotonin secretion from plate- 
lets. Increased vesicular transport has also been related to the formation 
of transendothelial channels. 5'+ » 89 jhese channels may be the result of the 
simultaneous opening of a chain of vesicles and may provide an avenue for 
nonspecific flux in hypertension. The presence of this channel is highly 
debated, however. 



24 

The nature of the hypertension itself can be a factor in increased 
capillary penneability.82 while acute hypertension is very likely to pro- 
duce a protein influx, chronic hypertension may protect the system in the 
likelihood of an acute rise in blood pressure. The reason for this is 
probably the hypertrophy of vascular muscles. This hypertrophy leads to 
an increased vascular resistance and thickening of the vessel wall pro- 
viding a mechanism for handling greater pressures. This is consistent with 
the observation that vasoconstriction decreases capillary permeability, 
while vasodilation increases this parameter. 

In some cases hypertension may lead to the rare malady, hypertensive 
encephalopathy. 21 This disease is related to the inability of the cerebral 
vasculature to acclimate to acute severe hypertension. In this instance 
cerebral edema occurs and this may cause swelling of the nervous tissue. 
This yields an increased cerebral pressure and may prevent vasodilation 
caused by a variety of stimuli. ''^ 

Hypervolemia may also result in an increase in protein extravasation.^ 
If mice, whose blood is about 1.3 ml, are treated systemically with high 
volumes (1 ml) of saline, extravasation of HRP can be documented. At 
lower volumes, however, no changes in permeability are observed. 

Since, in these experiments, the blood pressure increases only by 
20 mm Hg, the increased capillary permeability may not be due to hydro- 
static pressure. In order to open the BBB, pressure increases on the 
order of 100 mm Hg are required. 

The vasculature of primary brain tumors and of metastatic secondary 
brain cancers exhibits a generalized degradation. '^'^i >92 jh^s breakdown 
can be characterized by the presence of gap junctions, fenestra and open 
endothelial junctions, indicating the typical electron microscopic 



25 



structure of the tight junctions is destroyed. Although it has been sug- 
gested that these deficiencies should render cerebral tumors susceptible 
to treatment, 5 3 the clinical evidence does not support this. The therapy 
which has, thus far, been directed to brain neoplasms has been disappoint- 
ing at best. Neurosurgery may have reached its practical limit and most 
agree that major new advances must come from the field of chemotherapy.^^ 
It is interesting to note that many agents are effective against certain- 
peripheral tumors, but not their secondary brain metastases. 

The unresponsiveness of cerebral tumors has been explained by two 
phenomena. First, it is known that the greatest display of disintegration 
of the capillary structure occurs in the central, slow growing portion of 
the tumor, and as one moves to the periphery, the abnormalities decrease. 
This area of the tumor would be expected to show the highest resistance to 
chemotherapeutic agents. Secondly, since only a relatively small area of 
the brain vasculature is disturbed, any drug which reaches the neoplasm 
would rapidly diffuse into the outlying areas, thereby diminishing its 
concentration and effectiveness at the tumor site.'^^ Few suggestions have 
been forwarded to circumvent this problem. One involves osmotic opening 
of the BBB followed by methotrexate treatment, but this has a very limited 
usefulness because of problems with the accompanying edema. '^^ 

Many experimental models of brain injury are associated with an in- 
crease in capillary permeability. '^'".9'* These models include injury 
induced by dropped weights, pendulums, hammers, spring-mounted weights, 
blasting caps, rotary strikers, compressed air guns, humane stunners and 
accelerating devices. Generally, these procedures have been applied to 
the cat. 



26 



The leakage of proteins from the capillaries is usually proportional 
to the amount of injury, but even in severe injury the endothelial cell 
is intact and shows no sign of lesion. 5'* The explanation most often pro- 
posed for this extravasation is increased vesicular activity. In most in- 
juries there is an increase in serotonin and norepinephrine levels, as well 
as the production of a number of humoral agents. The increased vesicular 
content, which appears first in the arterioles and subsequently in the 
capillaries, may be mediated by stimulation of cAMP. There are also, how- 
ever, increases in blood pressure and that may act to increase vesicular 
transport. 

Cerebral infarcts also disrupt BBB function, as evidenced by an in- 
creased albumin concentration in the CSF of infarct victims. Correla- 
tion, however, between infarct size and location and the quantity of al- 
bumin leaked has not revealed any significance. 

Both electroconvulsive shock and pentylenetetrazole-induced seizures 
result in an increased capillary permeability to a number of plasma 
markers. ^»'*»^'* The degree of permeability increase is proportional to the 
number of shocks or compounds given. The morphological basis for this ex- 
travasation is not known and both junctional opening and increased vesi- 
cular transport have been suggested. It is interesting to note that, 
in both of these procedures, the blood pressure rises and this may be an 
important factor. 

Induced ischemia also produces a marked increase in the permeability 
of the cerebral vasculature. ^'^'^^ The experimental model which is most 
often used is that of the Mongolian gerbil. In this species, about half 
of the individuals lack arterial connections between cerebral and vertebral 
systems. After carotid occlusion and development of ischemia, HRP leaks 



27 

from the capillary lumen. In these investigations, there is no indication 
of endothelial cellular damage and, therefore, the extravasation of HRP is 
attributed to increased vesicular transport. The stimuli for this may be 
release of serotonin from platelets inducing vesicular formation secondary 
to vasoconstriction and increased blood pressure. Focal edema induced by 
a number of means, such as ultraviolet exposure, also increases vesicular 
transport and HRP uptake. 

Nonionizing radiation has long been implicated in BBB disruption. 
Microwaves and x-rays have been studied extensively in this regard. The 
effects of microwaves are highly controversial. One report states that 
exposure of rats to 2450 MHz at 10 mW/cm^ for two hours produces a marked 
extravasation. 56 This level is considered safe for human exposure in the 
United States, but not in other countries. The biological effects of 
microwaves can be subcl assified as changes resulting from gross thermal 
effects or nonthermal effects. In this study, the body temperatures of 
the animals were constant and the altered permeability of the BBB was 
attributed to microwave-stimulated serotonin release from platelets, 
although other mechanisms are possible. Conversely, a recent publication 
indicates that much higher levels of radiation are required to cause pro- 

9 7 

tein leakage, specifically 3000 mW/cm^. At these levels, cerebral tem- 
perature increases significantly and changes can be attributed to gross 
thermal effects. 

Porto-caval anastomosis, which causes severe liver dysfunction, has 
also been implicated in BBB breakdown. 5'*'^^ This disintegration has been 
termed hepatic encephalopathy. As in a myriad of other circumstances, 
increased vesicular transport has been implicated as the mechanism of 
extravasation. 



28 



Several situations involving autoimmune afflictions and induced auto- 
immunity, such as experimental allergic encephalomyelitis (EAE) which is 
used as a model of multiple sclerosis, demonstrate an increased capillary 
permeability. ^8 In EAE, the cerebral vessels are said to cuff or deform 
and this abnormality correlates with BBB breakdown. This may be important 
in the general progression of multiple sclerosis, as vascular changes are 
among the first changes which precede demyl el i nation. 

Agents which solubilize and fluidize membranes may also act to in- 
crease BBB permeability. 55'^°°'^°^ Dimethyl sulfoxide (DMSO), in very high 
concentrations, has been thought to increase the flux of such plasma mark- 
ers as inulin and mannitol. Its effects are said to be derived from its 
lytic action on membrane although interaction by micellular formation can- 
not be ruled out. Nortriptyline has a similar effect. Ethanol , in large 
doses, can also cause opening of the BBB to sucrose. It was postulated 
that this generalized increase in permeability may increase the suscepti- 
bility of the CNS to bacterial and viral infection. It has been shown, 
however, in acute and chronic doses of ethanol that were compatible with 
continued life, that there was no alteration in the BBB. ^° 2,10 3 

Many heavy metals have been associated in both general and specific 
changes in the BBB.i'56>ioi+ Mercury (II) Hg"*", in high concentrations, 
i.e. >80 ym, causes a generalized increase in the permeability of cerebral 
capillaries to sugars and protein markers but in low concentrations affects 
only the hexose carrier. Ionic lead has a similar effect. 

A number of conditions can alter BBB function in more subtle, yet no 
less damaging ways. In these instances, changes occur at the level of a 
specific carrier or carriers and, as such, only a particular type of com- 
pound is involved. These changes can result from physical alteration of 



29 

the carrier by denaturation. Additionally, diminution or increase of the 
blood level of a transportable compound can affect the utilization of the 
compound. In some cases, the rate limiting step in metabolism of a nutrient 
can be transferred from an enzyme to transit of the agent across the BBB.^^ 

Glucose transport is very important to CNS function. Under normal cir- 
cumstances, the rate-determining step in glucose metabolism involves the 
enzyme hexokinase. If, however, the plasma concentration of glucose falls, 
as in hypoglycemia, or cerebral metabolism increases, as in relative hypo- 
glycemia, the limiting step in utilization is shifted to transport of glu- 
cose across the BBB.^o 

In severe hypoxia (p02 <10 nm) , a number of progressive changes as- 
sociated with glucose flux occur. ^ In the dog, after one minute of oxygen 
deprivation, the influx of glucose into the brain is unchanged but efflux 
decreases. Ten minutes after initiation of hypoxia, influx and efflux of 
glucose decrease. This has been explained by a postulated modulator which, 
in the absence of hypoxia, is bound to the carrier and alters transport. 
In other animal models, different results are obtained. 

The transport of amino acids and, especially, neutral amino acids in 
certain disease states, has been the subject of much research. The neutral 
amino acid carrier is responsible for the transport of such neurotrans- 
mitter precursors as tryptophan, tyrosine and histidine and, as such, any 
interruption of this supply can have tremendous neurological consequences. 
Nowhere is this more apparent than in phenylketonuria. ^ '2° This syndrome, 
which is associated with high blood levels of phenylalanine, has been linked 
to mental retardation. This condition of high phenylalanine levels can 
act to competitively inhibit other substrates of this carrier, such as 
tyrosine, tryptophan, and histidine. This is made possible because of 



30 

the similar Km values for these amino acids in addition to the similarity 
between the Km's and plasma levels of these amino acids. Hyperphenylala- 
ninemia also can reduce protein synthesis by a similar mechanism. Treat- 
ment of this disease involves a phenylalanine restricted diet and 5-hydroxy- 
tryptophan supplements. 

In hepatic encephalopathy, the neutral amino acid carrier is induced 
and, as previously discussed, this induction may be related to increase in 
ammonia and glutamine levels. Dihydroxyphenyl alanine (L-DOPA), which is 
used as a dopamine source in the treatment of parkinsonism, is transported 
by the neutral amino acid carrier. It has been shown that ethanol increases 
DOPA transport as well as the transport of tyrosine, tryptophan and a- 
methyldopa into the CNS.io^ 

The monocarboxylic acid carrier appears to be a major organ for elim- 
inating metabolic acid wastes. As was previously discussed, the Km of 
lactate is close to its plasma concentration so that rises in lactate, 
such as those that accompany exercise, are only slowly translated to the 
CNS. In the case of anoxia, however, where cerebral levels of lactate 
rise, the systemic dissipation of this byproduct is slowed by the same 
phenomenon. 20 In the brain the loss of lactate is carrier and not diffusion- 
limited, unlike other organs. In hypoglycemia, lactate may act as an energy 
source. jhis is true in neonates, where lactate uptake is much higher 
than in adults. The Km of the monocarboxylic acid carrier is also corre- 
spondingly higher in neonates. 

Ketone bodies, which have an affinity for the monocarboxylic acid car- 
rier, are produced during fasting and can act as a metabolic energy source. 
These bodies include g-hydroxybutyric acid and acetoacetic acid. The en- 
zyme responsible for their production, g-hydroxybutyrate dehydrogenase, is 



31 



induced by starvation. The utilization of the surrogate food sources is 
limited by BBB transit. 

In starvation, the monocarboxylic acid carrier is said to be induced, 
although this is controversial. A recent paper denranstrated a lower Km 
and Vniax fo"" carriers in starved animals compared with control animals. ^3 
The increased flux of B-hydroxybutyrate and other substrates was attributed 
in this article to an increase in the diffusional component. 

A number of therapeutic agents have affinity for these carriers. These 
include such anionic compounds as probenicid, penicillin, and aspirin. 2° 
This affinity could potentially lead to competition and a decreased ability 
to eliminate metabolic acids from the CNS. It has been shown, however, 
that the concentrations required to cause inhibition are far above thera- 
peutic levels. 

The BBB is a complex system of enzymes, protein carriers, and vessels 
which impart to the brain a selective interface. This interface prevents 
potentially damaging substances from entering the brain without impeding 
the entry of nutrients or the exit or metabolites and excretory products. 
In adverse circumstances, the permeability of the barrier can be increased, 
resulting in a number of deleterious effects. 

Prodrugs and Drug Delivery Systems 

The BBB excludes a number of pharmacologically active agents and, as 
such, treatment of many cerebral diseases is severely limited. In order 
to increase the effectiveness of drugs which are active against central 
maladies, the pharmacokinetic profile of the agent must be augmented and, 
specifically, the transit time of the drug in the brain must be increased. 
If a method were available to implement these alterations, an agent could 
be delivered specifically to the brain. This specificity should increase 



32 



the therapeutic index of an agent since not only is the concentration of 
the agent increased in the vicinity of the bioreceptor but, of equal im- 
portance, the peripheral concentration of the drug is reduced decreasing 
any associated toxicity. 

Unfortunately, there are very few methods for circumventing the BBB 
and these are of limited usefulness. The direct administration of drugs 
into the CNS;i.e., an intrathecal injection has been used to deliver the 
folate antagonist, methotrexate, to the brain. This method is not very 
satisfactory since the distribution of methotrexate in the brain is uneven 
and slow.'*^ Additionally, since the ventricular volume of the CSF is 
small, increases in intracerebral pressure can occur with repeated injec- 
tions. This is particularly dangerous when the intracerebral pressure is 
already high as it is in CNS cancers. Repeated lumbar puncturing also 
carries a risk. 

A general method which can be applied to delivery of drugs to the 
brain is the prodrug approach. The term prodrug was coined by 
Albert and refers to the result of a transient chemical modification of a 
pharmacologically active agent. This change imparts to the compound an 
improvement in some deficient physiochemical property such as water solu- 
bility or membrane permeability. Ideally, a prodrug is biologically in- 
active but reverts to the parent compound in vivo . This transformation 
can be mediated by an enzyme or may occur chemically due to some designed 
instability in the agent. The aim of these manipulations is to increase 
the concentration of the active agent at its site of action and thereby, 
increase its efficacy. While potentially there are many different types 
of proderivatives, most thus far synthesized are simple esters and amides. 
These compounds are transformed to the parent acid, alcohol or amine by 
the ubiquitous hydrolases which are present in vivo . Many anticancer 



33 

agents have lent themselves to this type of manipulation. Several amides, 
for example, of the highly water soluble anticancer agent guanazole have 
been made. This series of lipoidal compounds hydrolyzed in vivo to yield 
the parent drug.^^^ A more sophisticated prodrug is cyclophosphamide, 
which is inactive in vitro . The agent is activated by P450 mixed function 
oxidases in the liver to the potent alkylating agent, N,N-bis(chloroethyl ) - 
phosphordiamidic acid.i^^ This drug is extensively used in cancer chemo- 
therapy. 

One of the most important applications of prodrugs is in the sustained 
release of therapeutically active agents. Cytosine arabinoside is used 
as an S-phase specific antimetabolite, but suffers from rapid metabolism 
by cytidine deaminase requiring a continual administration of the drug. 
This problem produced the prodrug cyclocytidine which is not a substrate 
for cytidine deaminase and which slowly releases the cytosine arabinoside 
by ring cleavage. 

The influx of a compound to any organ can be related by the equation 

Kp = QE 

where Kp is the clearance of a drug by an organ, Q is the blood flow 
through that organ and E is the extraction coefficient. extraction 
coefficient is related to the lipophil icity or octanol -water partition 
coefficient of a compound which is in turn related to the ability of a 
compound to partition into phosphol ipoidal membranes and consequently in- 
to organs. By increasing the lipophil icity of a compound with the pro- 
drug approach, one can increase the entry of a compound into its site of 
action but this is not specific and, in general, all organs are exposed 
to a greater tissue burden. Nonspecif icity is, therefore, one of the 
major drawbacks of the prodrug approach. This is especially important 



34 

with cytotoxic agents. While increased membrane permeability makes a 
compound more effective locally as a cytotoxic agent, there is almost 
always a disproportionate rise in systemic toxicity. This has severely 
restricted anticancer prodrugs of this type. 

Several types of toxicities are also associated with prodrugs. i^** 
Theoretically, a prodrug should be metabolized only to the parent compound 
but the formation of toxic metabolites by the prodrug is possible. This 
occurs with such compounds as phenacetin, a prodrug of acetaminophen. 
Another possible toxic reaction may be brought about by enzymatic or glu- 
tathione depletion. The compound thiamine tetrahydrofurfuryl disulfide, 
a prodrug of thiamine, requires glutathione-mediated disulfide bond cleav- 
age for activation and, therefore, toxicity may arise from the associated 
depletion of glutathione. 

The idea of using prodrugs to increase the specificity of delivery 
has been considered. This has proved, however, to be difficult and not 
very fruitful. Two basic approaches have been taken in this regard: site- 
directed or site-activated delivery and delivery by the association of a 
drug with a macromolecular carrier. 

The first method does not attempt to concentrate a compound at a par- 
ticular location, but is based on site-specific activation of the prodrug. 
For this to be possible, the enzyme responsible for the activation must 
be located specifically, or at least in high relative concentrations in a 
particular organ. The finding, for example, that y-glutamyl peptidase is 
present in high concentrations in the kidney led to a number of compounds 
substituted with the y-glutamyl group. Sul famethiazide and L-DOPA were 
derivatized in this manner in order to achieve renal delivery. 



This approach has been extensively applied to cancer chemotherapy . -^^^ 
The philosophy behind this application is related to the many differences 
that occur between cancer cells and normal cells. These variations are 
basically the result of the altered metabolism of neoplastic cells. Many 
alkylating agents have been synthesized in an attempt to capitalize on 
these differences. The lower pH of tumor cells has been exploited in a 
series of aziri dines which are more active at the pH of tumors than at 
physiological pH. The greater reducing power of cancerous sites has led 
to the development of a number of biologically inactive azo compounds 
which upon reduction yield potent cytotoxic agents. Examples of these 
are tetrazolium mustard and azomustard, both of which are reduced to aniline 
mustards. The inactivity of the parent compound is due to the delocaliza- 
tion of the nucleophilic nitrogen lone pair by the conjugated ring system. 

A number of 0-phosphate esters have been synthesized in order to take 
advantage of the high levels of acid phosphatase which are characteristic 
of human neopl asms . The 0-phosphate esters of p-hydroxy mustard and 
estradiol mustard were prepared as specific agents to be used in prostate 
cancers. The enzyme, y-glutamyl transpeptidase, is also found in high con- 
centrations in tumor cells so that y-glutamyl derivatives of cytosine ara- 
binoside and phenyl ene-di amine mustard have been proposed. The presence 
of hydrolytic esters has also been established in neoplastic formations. 
These include esterases and g-glucuronidases.^^^'^^^ The cytotoxic agent, 
aniline mustard, is converted in the liver as a result of a first pass 
effect to its 0-glucuronide. Tumors which contain high ^-glucuronidase 
activity convert the 0-glucuronide to the potent alkylating agent p-hydroxy- 
aniline mustard. 



36 



While these compounds have denranstrated some promise as anticancer 
drugs, for the most part they have not lived up to their potential. There 
are a number of reasons for this failing. The chemical manipulation of 
these agents may act to decrease their accessibility to a site of action. 
Additionally, if the agent is fairly lipophilic, it may "leak" from its 
site of action before exerting a pharmacological effect.^ in cancers 
there may also be diffusion limitations because of the restrictions in 
blood flow. 

A second approach for increasing the specificity of an agent for a 
particular organ involves the coupling of a pharmacologically active agent 
with a macromolecule.^^^ The specificity derived from such a system is 
related to the interaction between cells and endogenous and exogenous bio- 
polymers and macromolecules. Albumin, for example, is actively endocytosed 
by various macrophages . An anticancer compound could be coupled to al- 
bumin and the complex taken up by a macrophage tumor. This would then be 
directed to a lysosome where the drug would be hydrolyzed from its albumin 
carrier and exert its pharmacological action specifically. This has also 
been suggested as a means of treating DNA viruses which implant in macro- 
phages. Anthracyclines, such as daunorubicin and adriamycin, intercalate 
into DNA. A drug carrier has been devised in which fragments of DNA con- 
taining intercalated anthracyclines are administered systemically.^^'*'^^^ 
This intercalated complex is inactive but can be endocytosed and broken 
down in lysosomes by DNAases . Again, the aim is to release the cytotoxic 
agent in the vicinity of the malignancy and thereby increase its efficacy. 
Antibodies have also been studied as specific delivery carriers, but re- 
search has been hampered by the inhomogeneity of tumor-specific antibodies. 



37 



The idea of including a drug into liposomes formed in vitro has re- 
ceived a great deal of attention. ^ ^^'^ In these systems, the drug is 
inactive since it is enclosed in the phospholipoidal matrix of the lipo- 
some and, as in the case of the albumin conjugates, these packets are 
taken up by endocytosis. Again, the system should specifically exert its 
action at the site of influx. While these approaches are promising the- 
oretically, they have not met with much success. These carrier complexes 
are the subject of several recent books. ^^^'^^^ 

A general method was recently proposed for the specific delivery of 
drugs to the brain. This system was based on the results of some work 
with N-methylpyridinium-2-carbaldoxime chloride (2-PAM) . ,122,123 
This pyridinium quaternary compound is the agent of choice for the treat- 
ment of organophosphate poisonings, and exerts its action by reactivating 
deactivated chol inesterases . The problem with this agent is its highly 
polar nature. Organophosphates such as diisopropyl fluorophosphate (DFP) 
and paraoxon are very lipophilic and easily penetrate the BBB. The brain 
is, therefore, very susceptible to acetylcholinesterase inactivation by 
these agents. The highly polar 2-PAM has a very low activity in the brain 
since it is almost totally excluded by the BBB. To deal with this prob- 
lem the di hydro adduct of 2-PAM, pro-2-PAM,was synthesized as a prodrug. 




2-PAM pro-2-PAM 



38 

The rather involved synthesis of pro-2-PAM yielded the enamine salt 
which corresponds to the 3-protonated di hydro compound. The pK^ of the 
tertiary nitrogen in this enamine salt was determined potentiometrically 
and spectrophotometrically to be 6.32 ± 0.6 and the oxime was estimated 
to have a pK^ of -11. With a pKg, of 6.32, it would be predicted that 
about 90% of the enamine would exist as the free base at physiological 
pH (7.4). Since this dihydro free base is far more lipophilic than the 
parent quaternary, its ability to penetrate membranes is greatly enhanced. 
This pro-2-PAM is rapidly converted to the parent 2-PAM at physiological 
pH and the tj. has been determined to be 1.04 min by pharmacokinetic model- 
ing. This rapid conversion of the pro-2-PAM to 2-PAM is desirable since 
ideally, the only metabolism of the prodrug is to the parent compound. 

A number of experiments were carried out using 2-PAM and pro-2-PAM. 
From the linear descending portion of a semilog plot of blood concentra- 
tion versus time, the biological t,^ of 2-PAM and pro-2-PAM was calculated 
The ti for comparable doses of 2-PAM and pro-2-PAM differed by more than 

2 

60 min, and since the conversion of pro-2-PAM ^ 2-PAM is rapid, this dif- 
ference was assumed to be the altered distribution of pro-2-PAM. This in- 
dicates that even though the rate of conversion of pro-2-PAM is rapid, it 
is long enough for the enhanced distributional characteristics of the 
pro-2-PAM to be expressed. 

Blood levels of 2-PAM were significantly higher after an oral dose 
of pro-2-PAM than with a dose of 2-PAM. If pro-2-PAM is administered in- 
travenously, there are no new metabolites formed. If brain concentrations 
of 2-PAM are examined after 2-PAM and pro-2-PAM dosing, the concentration 
of 2-PAM in the brain is 13-fold higher in the case of pro-2-PAM admin- 
istration. If brain acetylcholinesterase is inactivated using DFP, the 



39 



extent of reactivation observed in mice injected with pro-2-PAM shows a 
dramatic increase over 2-PAM treated animals. 

A further experiment with 2-PAM showed that this small quaternary 
salt was rapidly lost from the CNS and this loss was attributed to an ac- 
tive efflux process. jhese results differ from those obtained by Ross 
and Froden who attempted to deliver a quaternary compound to the brain 
as its uncyclized cj-haloalkyl amine. ^^s ,126 jhey found that the loss of 
the quaternary compound was slow (tj^ = 38 hours) and concluded that the 
efflux of the quaternary was comparable to its influx. This difference 
in the rate efflux may be related to differences in molecular size, shape 
or charge. 

This preliminary work led to a proposed drug delivery system (Figure 
1-2) which is specific for the brain and which is generally applicable. ^^i* 
In this proposal, a pharmacologically active agent, whose ability to pass 
the BBB is low, is chemically linked to a pyridinium carrier. This car- 
rier could be envisioned as a nicotinamide or nicotinic acid ester. This 
complex would be reduced under conditions which would yield the dihydro- 
pyridine. This complex is then injected systemically and, because of the 
increased lipophil icity of the dihydropyri dine, partitions into the brain 
as well as into the periphery. In both locations, oxidation (kgx) should 
occur. The rate of this oxidation is somewhat controllable by the judi- 
cious placement of ring substituents on the pyridinium nucleus. Systemi- 
cally, the charged polar oxidized species should be eliminated rapidly 
by the kidney and/or liver i^Qy^iz^ > while in the brain the compound, be- 
cause of its charge and size, would be retained i.e., ^Q^^l2 ^ ''^outl " 

Also, in both locations, cleavage of the drug from its carrier should 
occur (kcieavage^- brain, the small nontoxic pyridinium carrier is 



40 




Figure 1-2. A Proposed Carrier-mediated Chemical Delivery System with 
Specificity for the Brain. The Drug Molecule to be Trans- 
ported is Represented by the (O). 



41 



rapidly eliminated, kgutS* cleavage of the drug from its carrier 

occurs at an appropriate rate i.e., k^ig^^^gg > k^^j^i a sustained release 
of the agent to the brain could also be obtained. Again, the cleaved 
polar drug would be rapidly eliminated systemically. By these manipula- 
tions a compound can be delivered specifically to the brain while its 
systemic concentration is kept low. This reduces any associated systemic 
toxicity of the agent and increases its therapeutic index dramatically. 

In this system the positively charged carrier complex cleaves to 
yield the active compound. The dihydropyridine is not, therefore, a pro- 
drug but rather a pro-prodrug or, better stated, a chemical delivery sys- 
tem. This drug delivery system is based on the naturally occurring re- 
duced nicotinamide adenine dinucleotide (NADH) t oxidized nicotinamide 
(NAD''') system. These endogenous coenzymes are important in electron trans- 
ferring chains and their suitability to this purpose is related to the 
chemistry of dihydropyri dines and enamines. In the described delivery 
system the relative unstability, greater 1 ipophil icity and predictability 
of chemistry of the dihydromoieties are exploited. Additionally, since 
this method relies on enzymatic activation by an endogenous system (NADH 
dehydrogenase) whose substrates closely approximate the delivery compounds, 
any toxicity associated with the oxidation should be minimal. In this 
system the BBB has not been simply circumvented but rather used as an in- 
tegral part of the delivery scheme. 

Statement of the Problem 

The chemical delivery system proposed by Bodor et al . should demon- 
strate a broad applicability since the agent to be del ivered is simply 
attached to a particular carrier. ^^'^ In certain instances, however, this 
system can be simplified. If a pyridinium nucleus isanintegral structural 



42 

component of a molecule, as it is in many phamacologically active agents, 
the molecule is provided with an internal delivery moiety. The drug to 
be delivered and the carrier are therefore merged into one molecule. The 
pharmacokinetics of this scheme is also simpler than those involved with 
the carrier system since no cleavage is necessary of the delivered mole- 
cule from the carrier. In this approach, which appears in Figure 1-3, 
an appropriately chosen pharmacologically active compound i.e., one which 
contains a pyridinium moiety, would be reduced to its corresponding di- 
hydropyridine. This lipophilic species would penetrate the BBB as well 
as into the systemic circulation. After a period of time determined by 
the stability of the compound, it would be oxidized to the parent quater- 
nary salt (kox)- Systemically, this agent would rapidly be eliminated 
by filtration or by tubular secretory mechanisms (kQ^tz)- I" 
however, since the ability of the compound to freely diffuse would be lost, 
it would be delivered fairly specifically (kQ^J^2 " "^outl^" transit 
time of the drug in the brain would depend upon a number of factors in- 
cluding the participation of the compound in any active efflux processes. 
It would be hoped that in most cases the rate at which the compound entered 
the brain would be faster and ideally, much faster than the rate at which 
the compound left the brain (kQ^^-i). 

This system, unlike the carrier mediated chemical delivery system, 
can be considered a prodrug. This prodrug should, however, demonstrate 
a specificity for the brain because of its design, the characteristics 
of the BBB, and the chemistry of dihydropyridines . Again, this specificity 
should increase the therapeutic index of the drug delivered. 

Several criteria were used in choosing a molecule for these delivery 
approaches. Obviously, the compound should contain a pyridinium moiety 



43 



SEE. 




Figure 1-3. The Proposed Drug Delivery System. 

1 



44 



which is reducible to some dihydropyridine species which is stable enough 
to be isolated. The compound should be active in vitro . Since quaternary 
compounds are to be considered, the lack of any in vivo activity might be 
ascribed to transport or distributional problems. A review of the litera- 
ture showed two groups of compounds which looked particularly suitable. 
These include the substituted benzophenanthridinium salts and the proto- 
berbine alkaloids which have the basic skeleton: 





benzophenanthridinium ion 



protoberbine 



Members of these groups contain an N-substituted isoquinoline moiety 
which can be reduced by a number of agents. A number of these dihydroiso- 
quinoline compounds are stable. ^^7 jhese agents show a wide range of 
effects including antineoplastic and antibiotic activity. ^^s-ue j^g 
specific compound chosen was berberine. 




OCH3 

berberine 

Berberine, which has the chemical name 5,6-dihydro-9,10-dimethoxybenz- 
[g]-[l .3]benzodioxolo[5,6-a]quinolizinium chloride, has a rather high 
in vitro activity against several cancer types including Ehrlich and lym- 
phoma ascites. ^31, 133 j^s in vivo action is, however, very low. ^^2, 133 



45 



Berberine is widely distributed in the plant kingdom and is found 
in such families as menispennaceae and berberidaceae, to name only two.^^'' 
The compound was isolated by 1826 by Pelletan and Chevallier. 

Biosynthetically, berberine is interesting because of the presence 
of the so-called berberine bridge. This term refers to the single carbon 
atom between the nitrogen and the methoxylated aromatic nucleus. Two post 
ulates for the formation of this have been suggested. The first involves 
formaldehyde in a Mannich-type ring closure while the second involves an 
oxidative cyclization of an N-methyl group. -^^^'^^^ Labelling experiments 
favor the second mechanism.^ 

There are several total syntheses for berberine in the literature 
and the chemistry of the protoberbine alkaloids is well reviewed. 
Berberine has a variety of pharmacological actions and several therapeutic 
uses. Pharmacologically, it exhibits a depressive action on excitable 
tissues, ^"^^ induces hypotension and tachycardia,^'*'*'^'*^ and inhibits a 
number of enzymes including histaminease (human pregnancy plasma diamine 
oxidase) chol inesterase,^'^'* dopamine- adenyl cyclase,^'*'' and cation- 
dependent ATP phosphorylases.^'*^ Berberine also possesses an antiheparin^ 
and local anesthetic activity.^'*'* Because of the affinity of berberine 
for dopaminergic receptors and alcohol dehydrogenases it has been 
used to characterize geometric and stereospecific requirements for sub- 
strate binding to these enzymes. Berberine has long been known as an anti 
biotic. The alkaloid causes mutations in certain bacterium by affecting 
nonchromosomal genetic material .^^'^ Berberine has been used mostly in 
India to treat cholera diarrhea,^ leshmaniasis and other parasitic 
infections . 



46 

Biochemically, berberine acts to inhibit DNA, RNA and protein syn- 
thesis. ^"^^ II has been suggested that many compounds which are structur- 
ally similar to berberine exert their cytotoxic activity via alkylation 
at the iminium site.^^s /\ ^^jre thoroughly studied, and perhaps more 
accurate, hypothesis is that berberine acts by intercalating between the 
base pairs of DNA. This intercalation can be explained by a modified 
neighbor-exclusion model. ^^'^ A model which has been used to explain the 
intercalation of berberine is one in which the greater portion of rings A, 
B and D are intercalated. As berberine complexes, it assumes a more 
rigid planar species as may be indicated by the increase in fluorescence 
quantum yield. This intercalation does, however, slightly bend the double 
helix. The unwinding angle of DNA due to intercalation of berberine is 
less than for the more planar molecules such as corylene. The effect of 
the positive charge on intercalation has also been noted. 

Berberine, therefore, is appropriately suited as a candidate for in- 
clusion in the drug delivery system described. In order to verify the 
original hypothesis of site-specific delivery, a number of experiments 
are required. Dihydroberberine must be synthesized and its stability 
assayed. The ability of dihydroberberine to penetrate the BBB and concen- 
trate in the brain must also be demonstrated. Additionally, the toxicity 
and anticancer activity of the agent should be investigated. 



1 

I 



I 



CHAPTER 2 
MATERIALS AND METHODS 
Elemental analyses of compounds synthesized were performed by Gal- 
braith Laboratories, Inc., Knoxville, Tennesse, or Atlantic Microlab, 
Inc., Atlanta, Georgia. Uncorrected melting points were determined using 
a Thomas-Hoover melting point apparatus. Ultraviolet spectra (UV) were 
recorded on a Beckman 25, Gary 210 or Gary 219 spectrophotometer. An 
Apple II Plus microprocessor was dedicated to the Gary 210 instrument. 
Infrared spectra (IR) were taken on a Beckman 4210 high resolution and a 
Beckman Acculab 1 infrared spectrophotometer. Samples were analyzed as 
a thin film between sodium chloride windows or as a potassium bromide pel- 
let. Nuclear magnetic resonance spectra (NMR) were obtained from either 
a Varian T60 or Joel-JNM-FX 100 Fourier transform spectrometer. The sam- 
ples were dissolved in deute rated chloroform (GDGI3), deuterated di methyl - 
sulfoxide ((GDJ.SO), deuterated pyridine (G5D5N), deuterated methanol 
(GD3OD), deuterated acetonitrile (GD3GN), deuterium oxide {D2O) or tri- 
fluoroacetic acid (TFA). Ghemical shifts in parts per million were re- 
ported relative to the internal standard tetramethylsilane except in aque- 
ous systems where sodium 3-tri methyl si lylpropanesulfonate is used. Mass 
spectra were obtained using a DuPont 21-491B double focusing magnetic sec- 
tor mass spectrometer to which was dedicated a Hewlett Packard 2100A com- 
puter. In all determinations, the ionizing voltage was 70 eV. 

High pressure liquid chromatography (HPLG) was performed on either 
a Waters Associates system consisting of a Model U6K injector, a Model 
6000A solvent delivery system and a Model 440 absorbance detector or a 

47 



48 

ternary Beckman system consisting of three Model 112 solvent delivery 
systems, a Model 160 absorbance detector and a Model 421 controller. In 
some studies, a Varian Fl uorichrom'^ detector was used with one of the 
Beckman pumps. Thin-layer chromatography (TLC) was performed on EM Re- 
agents Cat. 5751 Aluminum Oxide 60 F-254 precoated plates (layer thickness 
0.25 mm) or Analtech, Inc. Uniplate'^, which are precoated with silica gel 
G at a thickness of 0.25 mm. Tissues were homogenized by a VirTis 45 
homogenizer or by a teflon pestle and ground glass tube. In potentiomet- 
ric titrations, a Radiometer-Copenhagen PHM 84 Research pH meter was used. 
A Sage Instrument Co. Model 341 A syringe pump was used for infusions. 
For radionuclide studies, a Packard Tri-Carb 460 CD Liquid Scintillation 
system was employed. 

In the theoretical studies, the MINDO/3 program was modified to suit 
the University of Florida's IBM System/370 computer. The molecule draw- 
ing program, X3DM0L, was developed by E.W. Phillips. 

All chemicals used were of reagent grade. Berberine was obtained 
from Sigma Co. while nicotinic acid, nicotinamide, methyl iodide, benzyl 
bromide, butanol , hexanol , octanol , decanol , sodium borohydride and sodium 
dithionite were obtained from Aldrich Co. Phenethyl alcohol was purchased 
from Matheson, Coleman and Bell. Tritiated inulin, Soluene'^ and scintilla- 
tion cocktails were obtained from New England Nuclear. Solvents were of 
chromatographic purity and were obtained from Fisher Co. Pyridine (Aldrich 
Co.) was refluxed over CaH2 and distilled before using. In cases where 
oxygen was to be excluded, solutions were made from water which, after 
boiling for fifteen minutes, was cooled with a stream of pyrogallol- 
scrubbed nitrogen passing through it. A phosphate buffer (pH 7) was made 
by dissolving 1.183 g of potassium dihydrogen phosphate and 4.320 g of 
di sodium hydrogen phosphate in water and diluting to 1.0 i. 



49 



Synthesis 

Dihydroberberine (2) 

Seven grams of berberine chloride (1) were dried over phosphorous 
pentoxide at 80°C in a vacuum oven for eight hours. Five grams of the 
dried salt (0.013 moles) were added to a suspension of dry pyridine and 
0.6 g of sodium borohydride (NaBHii). The solution was stirred under N2 
for twenty minutes at room temperature, at which time 0.5 g more of 
NaBHi^ were added. The liquid was then poured into 800 ml of ice water. 
The ensuing precipitate was dried overnight at 50°C over phosphorous pen- 
toxide. The crude material was recrystallized from benzene-petroleum 
ether (low boiling). The yield was 35%: Melting point 156-158°C, Litera- 
ture 157-159°C; NMR (CDCI3) 5 7.1 (1 H, s). 6.7 (2 H, s), 6.4 (1 H, s), 
5.9 (3 H, s), 4.3 (2 H, s), 3.4 (6 H, s), 3.1 (2 H, t), 2.9 (2 H, t) ; 
MS m/e 338 (M+ + 1) 22%, 337 (M"^) 100%, 322 (M"^ - 15) 27%, 278 (M"^ - 59) 
13%; UV (95% ethanol) 370 IR (KBr) v (C-H) 3005 and 2970, (C-C, C-N) 

1600 and 1482, (C-O-C asym) 1227 and 1270, (C-O-C sym) 1028 and 1083; 

NMR (see figure 3-6); Elemental analysis calculated %: C, 71.21; 
H, 5.60; N, 4.15. Found %: C, 71.19; H, 5.70; N, 4.14. 
Dihydroberberine Hydrochloride (3) 

One gram of (2) was dissolved in a minimal amount of CH2CI2. Anhy- 
drous hydrogen chloride, produced by dropping concentrated sulfuric acid 
(H2S0i^) on sodium chloride (NaCl), was bubbled through the solution yield- 
ing a yellow precipitate. The material was recrystallized in aqueous 
ethanol . 

9-Demethyl berberine (Berberrubin) (4) 

Seven grams of (1) were dried for two hours at 100°C in a vacuum oven 
and then were heated to 200°C for an additional thirty minutes. The deep 
purple material produced was dissolved in hot water and extracted with 



< 



50 



chloroform (CHC13). The organic layer was reduced to dryness and the 
residue dissolved in hot water. The solution was filtered and then made 
acidic with an excess of hydrochloric acid (HCl). The ensuing precipi- 
tate (yellow-brown solid) was collected by filtration and redissolved in 
hot water. The solution was filtered and made basic with potassium hy- 
droxide. The solution turned deep purple and crystallization was induced 
by scratching: NMR (CD3OD) 6 9.0 (s, 1 H), 7.8 (s, 1 H), 7.2 (m, 2 H), 
6.8 (m, 2 H), 5.8 (s, 2 H), 3.7 (s, 3 H), 4.4 (m, 2 H), 3.0 (m, 2 H); 

UV (95% ethanol) 276 nm and 238 nm a others 512 nm; IR (KBr) v (C-H) 

max 

3000 and 2900, (C-C, C-N) 1635, 1571, 1509 and 1472, (C-O-C asym) 1221 
and 1289, (C-O-C sym) 1035 and 1144; Elemental analysis calculated %: 
C, 71.03; H, 4.67; N, 4.36. Found %\ C, 71.20; H, 4.81; N, 4.42. 
Berberine Iodide (5) 

A solution of 1.0 g of (4) in acetone was prepared. A 1.0 M excess 
of methyl iodide was added and the solution was allowed to reflux for 
several hours. The characteristic yellow color of berberine appeared and 
TLC, NMR, IR, and UV confirmed the methyl ation. 

3-(Aminocarbonyl )-l-methylpyridinium Iodide/1 -Methylnicotinamide Iodide (6)^ 

Five grams of nicotinamide (0.041 moles) were dissolved in 50 ml of 
dry methanol. A molar excess of methyl iodide (11.6 g) was added to the 
stirring mixture and after one hour of refluxing, a precipitate formed. 
This was filtered and washed. The material was recrystall ized from aque- 
ous methanol: Melting point 101-103°C, Literature 102-105°C; UV (H2O) 
224 mm and 265 mm A_-„; NMR and IR were identical with the literature. 



*The nicotinic acid derivatives are given a systematic name followed by 
a common name 



51 



3-(Aminocarbony"l )-1-( phenyl methyl )pyridinium Bromide/1 -Benzyl nicotinamide 
Bromide (ij 

Ten grams of nicotinamide (0.083 moles) were dissolved in 150 ml of 
methanol. A molar excess of benzyl bromide (28.0 g) was added and the 
mixture was allowed to reflux for several hours. Upon cooling, a white 
solid appeared. This was filtered, washed and recrystallized from metha- 
nol: Melting point 206-208°C, Literature 205°C; NMR (D2O or TFA) 
5 9.5 (1 H, s), 9.0 (2 H, m) , 8.2 (1 H, m) , 7.5 (5 H, s), 5.9 (2 H, s); 
IR (NaCl) V (N-H asym) 3300, (N-H sym) 3148, (-C(=0)NH2 Amide I) 1690, 
(Amide II) 1643, (C-N-C) 1388. 

3-Pyridinecarboxylic Acid Ethyl Ester/Ethyl Nicotinate (8) 

Forty- two grams (0.34 moles) of nicotinic acid were mixed with 55 ml 
of absolute ethanol and 25 ml of concentrated H^SO^. The mixture was 
refluxed in an oil bath for four hours at which time the solution was 
poured over ice and made slightly basic with ammonia. The aqueous solu- 
tion was extracted with ethyl ether. The organic layer was dried with 
sodium sulfate (Na2S0i+) and the solvent evaporated under reduced pressure. 
The product was a clear liquid and the yield was 55%: NMR (CDCI3) 6 9.2 
(1 H, s), 8.8 (1 H, m), 8.2 (1 H, m) , 7.4 (1 H, m) , 4.4 (2 H, q), 1.4 
(3 H, t); IR (NaCl) v (C-H) 2990, (C=0) 1728, (-C-C(=0)0) 1286, (0-C-C) 
1112, (y CH) 742. (3 ring) 703. 

3-Pyridinecarboxylic Acid Butyl Ester/Butyl Nicotinate (9) 

Forty-two grams (0.34 moles) of nicotinic acid were dissolved in 
55 ml of 1-butanol and 25 ml of concentrated HjSO^ was slowly added. The 
solution was heated to reflux in an oil bath for three hours, at which 
time the solution was poured over ice and made slightly basic with ammonia. 
The mixture was extracted with ethyl ether. The separated ether layer 
was dried over Na2S04 and the solvent removed under reduced pressure. 



52 

The product was a clear liquid and the yield was 62%: NMR (CDCI3) 

9.2 (1 H, s), 8.8 (1 H, m), 8.2 (1 H, m) , 7.4 (1 H, m) , 4.3 (2 H, t) , 

1.6 (4 H, m), 0.97 (3 H, t); IR (NaCl ) v (C-H) 2962, (C=0) 1730, (C-C, 

C-N) 1594, (-C-C(=0)0) 1288, (0-C-C) 1117, (y CH) 742, (b ring) 702. 

3-Pyridinecarbonyl chloride Hydrochloride/Nicotinoyl Chloride Hydrochloride 
(10) 

Forty-one grams (0.33 moles) of nicotinic acid were stirred in an ice 
bath with 110 ml of thionyl chloride (SOCI2) slowly added. After the 
addition was complete, the mixture was refluxed for three hours. The 
S0C12 was removed under reduced pressure and traces of SOCI2 were azeo- 
troped off with benzene. The white crystalline product was obtained in 
94% yield; NMR and IR were identical with the literature. 
3-Pyridinecarboxylic Acid Hexyl Ester/Hexyl Nicotinate (11) 

Ten grams of (10) (0.062 moles) were dissolved in 100 ml of dry dis- 
tilled pyridine and 5.73 ml (0.062 moles) of 1-hexanol. The solution was 
refluxed in an oil bath for six hours. The solution was then poured over 
ice which had been made basic with ammonia. The aqueous solution was ex- 
tracted with ethyl ether, the organic layer dried over Na2S0^, and the 
solvent removed under reduced pressure. The yield was 60%: NMR (CDCI3) 
6 9.1 (1 H, s), 8.7 (1 H, m), 8.2 (1 H, m) , 7.3 (1 H, m) , 4.3 (2 H, t) , 
1.2 (8 H, m), 0.87 (3 H, t); IR (NaCl) v (C-H) 2938 and 2961, (C=0) 1726, 
(C-C, C-N) 1590, (-C-C(=0)0) 1282, (0-C-C) 1111, (y CH) 741, (g ring) 702. 
3-Pyridinecarboxylic Acid Octyl Ester/Octyl Nicotinate (12) 

Ten grams of (10) (0.062 moles) were dissolved in 100 ml of dry pyri- 
dine and 9.75 ml (0.062 moles) of 1-octanol. The solution was heated to 
reflux in an oil bath and the progress of the reaction monitored with TLC. 
After eight hours, the liquid was poured over ice which had been made basic 
with ammonia. The aqueous solution was extracted with ethyl ether. The 



53 

ether layer was dried over sodium sulfate (Na2S0i^) and the solvent removed 
under reduced pressure. The yield was 58%: NMR (CDCI3) 6 9.1 (1 H, s), 
8.7 (1 H, m), 8.2 (1 H, m), 7.3 (1 H, m), 4.3 (2 H, t), 1.3 (12 H, m) , 
0.88 (3 H, t); IR (NaCl ) v (C-H) 2920 and 2950, (C=0) 1723, (C-C, C-N) 
1589, (-C-C(=0)0) 1277, (0-C-C) 1109, (y CH) 732, (g ring) 693. 
3-Pyridinecarboxylic Acid Decyl Ester/Decyl Nicotinate (13) 

Ten grams of (10) (0.062 moles) were dissolved in 100 ml of dry pyri- 
dine and 11.82 ml (0.062 moles) of 1-decanol. The solution was refluxed in 
an oil bath for eight hours. The liquid was then poured over ice which 
had been made basic with ammonia. The aqueous solution was extracted with 
ethyl ether, the organic layer dried over Na2S04, and the solvent removed 
under reduced pressure. The yield was 64%: NMR (CDCI3) 6 9.2 (1 H, s), 
8.7 (1 H, m), 8.2 (1 H, m), 7.2 (1 H, m), 4.3 (2 H, t), 1.2 (16 H, m) , 
0.83 (3 H, t); IR (NaCl) v (C-H) 2915 and 2946, (C=0) 1720, (C-C, C-N) 1584, 
(.C-C(=0)0) 1275, (0-C-C) 1105, (y CH) 732, (b ring) 692. 
3-Pyridinecarboxylic Acid B-Phenylethyl Ester/ B-Phenethyl Nicotinate (14) 

Twenty-six grams (0.15 moles) of (10) were dissolved in 200 ml of 
pyridine. To this stirring solution was added dropwise 18.3 g (0.15 moles) 
of B-phenethyl alcohol. The solution was refluxed in an oil bath for 
several hours. The solution was then poured over ice and made slightly 
basic with ammonia. This solution was then extracted with ether. The 
organic layer was dried over Na2S0i+ and evaporated under reduced pressure. 
The yield was 62%: ^H NMR (CDCI3) 6 9.1 (1 H, s), 8.6 (1 H, m) , 8.0 (1 H, m), 
7.2 (6 H, s), 4.4 (2 H, t) , 2.9 (2 H, t) ; IR (NaCl) v (C-H) 3030 and 2959, 
(C=0) 1722, (C-C, C-N) 1585, (-C-C(=0)0) 1276, (0-C-C) d 1120, 1105, (y CH) 
737, (b ring) 695. 



54 

3- (Ethoxycarbonyl)-I -methyl pyridinium Iodide/Ethyl M - Methyl nicotinate 
Iodide (T5) 

Ten grams of (8) (0.066 moles) were mixed with methanol and with a 
molar excess of methyl iodide (18.7 g). The solution was refluxed for 
several hours. The solvent was removed, yielding a red-orange oil which 
solidified on cooling to a yellow solid. The solid was recrystall ized 
from acetone-ether: NMR (CDCI3, (003)^50), 6 9.6 (2 H, m) , 9.0 (1 H, m] 
8.4 (1 H, m), 4.8 (3 H, s), 4.5 (2 H, q), 1.4 (3 H, t); IR (NaCl) v (C-H) 
3008, (C=0) 1723, (-C-C(=0)0) 1303, (0-C-C) d 1102, 1117, (y CH ) 742, 
(6 ring) 654. 

3-(Butoxycarbonyl )-l-methylpyridinium Iodide/Butyl N - Methyl nicotinate 
Iodide (16) 

Ten grams of (9) (0.056 moles) were dissolved in 60 ml of acetone 
and a molar excess of methyl iodide (15.9 g) was added. The solution was 
refluxed for two hours. The solvent was removed, leaving a yellow solid 
which was recrystall ized from acetone-ether: NMR (CDCI3, (003)^80) 
6 9.9 (2 H, m), 9.0 (1 H, m), 8.6 (1 H, m), 4.5 (3 H, s), 4.4 (2 H, t) , 
0.93 (3 H, t); IR (NaCl) v (C-H) 2950, (C=0) 1720, (-C-C(=0)0) 1291, 
(0-C-C) 1100, (y CH) 735, (b ring) 651. 

3- (Hexoxycarbonyl)-l -methyl pyridinium lodide/Hexyl N - Methyl nicotinate 
Iodide (17) 

Ten grams of (11) (0.048 moles) were dissolved in 60 ml of acetone, 
and a molar excess of methyl iodide (13.7 g) was added. The solution was 
refluxed for two hours. The solvent was removed, producing an orange oil 
iH NMR (CDCI3, (003)^50) 6 9.3 (2 H, m), 8.9 (1 H, m) , 8.3 (1 H, m), 
4.7 (3 H, s), 4.3 (2 H, t), 0.90 (3 H, t) ; IR (NaCl) v (C-H) 2944, (C=0) 
1723, (-C-C(=0)0) 1295, (0-C-C) 1111, (y CH) 738, (g ring) 654. 



55 



3- (Octoxycarbonyl )-1 -methyl pyridinium lodide/Qctyl N - Methyl nicotinate 
Iodide (18) 

Ten grams of (12) (0.043 moles) were mixed with 60 ml of acetone and 
with a molar excess of methyl iodide (12.1 g). The solution was allowed 
to reflux for two hours at which time the solvent was removed, yielding an 
oil which was resistant to crystallization: NMR (CDCI3, (003)^50) 6 9.9 
(2 H, m), 9.0 (1 H, m), 8.6 (1 H, m), 4.6 (3 H, s), 4.4 (2 H, t), 0.92 (3 H, 
t); IR (NaCl) v (C-H) 2960, (C=0) 1731, (-C-C(=0)0) 1302, (0-C-C) 1120, 
(y CH) 749, (B ring) 668. 

3- ( Decoxycarbonyl )-l -methyl pyridinium lodide/Decyl N- Methyl nicotinate 
Iodide (19) 

Ten grams of (13) (0.038 moles) were dissolved in 60 ml of acetone 

and a molar excess of methyl iodide (10.8 g) was added. The solution was 

refluxed for two hours. The solvent was removed, leaving an orange oil: 

iH NMR (CDCI3, (003)^50) 6 9.7 (2 H, m) , 9.0 (1 H, m) , 8.5 (1 H, m) , 4.8 

(3 H, s), 4.4 (2 H, t), 0.95 (3 H, t); IR (NaCl) v (C-H) 2944, (C=0) 1725, 

(-C-C(=0)0) 1297, (0-C-C) 1113, (y CH) 741, (b ring) 658. 

3-(g-Pheny1ethoxycarbonyl )-l -methyl pyridinium lodide/g-Phenethyl N-Methyl- 
nicotinate Iodide (20) 

Ten grams of (14) (0.044 moles) were dissolved in 60 ml of acetone. 
A 1.0 M excess of methyl iodide (12.5 g) was added to the liquid and the 
system was allowed to reflux for two hours. The solvent was removed, 
yielding a solid which was recrystallized from acetone: NMR (CDCI3, 
(CD,) SO) 5 9.4 (2 H, m), 8.7 (1 H, m) , 8.2 (1 H, m) , 7.2 (5 H, s), 4.7 
(3 H, s),4.6(2 H, t), 3.1 (2 H, t); IR (NaCl) v (C-H) 2995 and 3023, 
(C=0) 1724, (-C-C(=0)0) 1290, (0-C-C) d 1135 and 1124, (y CH) 731, (6 ring) 
657. 



56 

1 ,4-Dihydro-1 -methyl -3-pyridinecarboxamide/1 -Methyl -1 ,4-Dihydronicotin- 
amide (21) 

Four and six- tenths grams of sodium hydrogen carbonate (NaHCOg) and 
2.64 g of (6) (0.019 moles) were dissolved in 100 ml of water and cooled 
in an ice bath. To this stirring solution was added 6.96 grams of sodium 
dithionite (Na2S20j^). A stream of nitrogen (No) covered the reaction mix- 
ture. After one hour, the reaction was stopped and the solution was ex- 
tracted with several aliquots of CHCI3. The CHCI3 layer was removed under 
reduced pressure, yielding an orange oil. The oil was dissolved in a min- 
imal amount of CHCI3 and tritrated with petroleum ether. From this, an 
oil appeared and this was removed and dried in vacuo : NMR (D^O) 6 6.9 
(1 H, s), 5.7 (1 H, d), 4.8 (1 H, m), 3.2 (2 H, s), 3.0 (3 H, s); UV 355 nm 

x_^; Elemental analysis calculated %: C, 60.87; H, 7.25; N, 20.20. Found 
max 

%: C, 60.92; H, 7.29; N, 20.36 (C7H^oN20). 

1 .4-Dihydro-l-( phenyl methyl )-3-pyridinecarboxamide/l-Benzyl-l ,4-dihydro- 
nicotinamide (22)" 

To 100 ml of water were added 4.6 g of NaHCOs and 2.93 g of (7) (0.013 

moles). The solution was cooled and 6.96 g of Na2S20^ were added. After 

two hours of stirring under N2, a precipitate formed. The solution was 

filtered. The solid was recrystall ized from aqueous methanol, giving 

lemon-yellow needles: NMR (D2O) 6 7.2 (6 H, s), 7.1 (1 H, s), 5.7 (1 H, 

m), 4.7 (1 H, m), 4.2 (2 H, s), 3.1 (2 H, m) , in CDCl 3 two protons at 5.86 

appear; UV 357 nm \ ; Elemental analysis calculated %: C, 72.29; H, 6.58; 
'^^ max 

N, 12.97. Found %: C, 72.09; H, 6.60; N, 12.84 (Ci3H^^N20). 

1 ,4-Dihydro-l-methyl-3-pyridinecarboxylic Acid Ethyl Ester/Ethyl 1,4-Di- 
hydro-N-methylnicotinate (23) 

Four and six-tenths grams of NaHCOs and 2.75 g of (15) (0.016 moles) 
were dissolved in 100 ml of water and cooled in an ice bath. To this stir- 
ring solution was slowly added 6.96 g of Na2S20^. Two hundred milliliters 



57 

of ethyl ether were then added so that the di hydro would be extracted 
upon formation. This two-phase system avoided tetrahydropyridine produc- 
tion. The reaction proceeded for one hour under nitrogen. The ether layer 
was removed and the aqueous layer extracted. The combined ether fractions 
were dried over Na2S0^ and the solvent removed under reduced pressure. The 
resulting orange-red oil was dried in vacuo : NMR (CDCl^) 6 6.9 (1 H, d) , 
5.6 (1 H, m), 4.8 (1 H, m) , 4.1 (2 H, g), 3.1 (2 H, m), 2.9 (3 H, s), 1.2 
(3 H, t); UV 358 nm x^^^; Elemental analysis calculated %: C, 64.67; H, 8.17; 
N, 8.43. Found %: C, 64.65; H, 7.88; N, 8.34 (C9H13NO2). 

1 ,4-Dihydro-l-methyl-3-pyridinecarboxylic Acid Butyl Ester/Butyl 1 ,4-Dihy- 
dro-N-methylnicotinate (24) 

A solution of 4.6 g of NaHCOj and 3.12 g of (16) (0.016 moles) was 
prepared in 100 ml of water. The solution was cooled and 6.96 g of Na2S20it 
were added. Two hundred milliliters of ethyl ether were added and this 
mixture stirred under nitrogen for one hour. The ether layer was removed, 
dried with Na2S0i+ and reduced in volume. The resulting oil was dried in 
vacuo: NMR (CDCI3) 6 6.9 (1 H, s), 5.6 (1 H, m), 4.7 (1 H, m) , 4.1 
(2 H, t), 3.1 (2 H, m), 2.9 (3 H, s), 0.9 (3 H, t); UV 358 nm x^^; Ele- 
mental analysis calculated %: C, 67.69; H, 9.07; N, 7.23. Found %: C, 
67.58; H, 8.82; N, 7.09 (C11H17NO2). 

1 ,4-Dihydro-l -methyl -3-pyridinecarboxylic Acid Hexyl Ester/Hexyl 1,4- 
Di hydro-N-methyl ni coti nate (25 ) 

A solution of 4.6 g of NaHCOj and 3.57 g of (17) (0.016 moles) was 
prepared in 100 ml of water. The solution was cooled and 6.96 g of 
Na2S20^^ were added. Two hundred milliliters of ethyl ether were added 
and the mixture stirred under nitrogen for one hour. The ether layer was 
separated, dried and reduced in volume. The dried oil was orange in color: 
iH NMR (CDCI3) 6 6.8 (1 H, s), 5.5 (1 H, m) , 4.6 (1 H, m) , 4.0 (2 H, t). 



58 

3.1 (2 H, m), 3.0 (3 H, s), 0.9 (3 H, t) ; UV 359 nm x^^^; Elemental analy- 
sis calculated %: C, 69.96; H, 9.73; N, 6.32. Found %: C, 69.82; H, 9.46; 
N, 6.28 (C^gH^^NOj. 

1 ,4-Dihydro-l -methyl -3-pyndinecarboxylic Acid Qct.yl Ester/Oct.yl 1,4-Dih y- 
dro-N-methylnicotinate (267 ~~ 

A solution of 4.6 g of NaHCOs and 4.02 g of (18) (0.016 moles) was 
prepared in 5% aqueous methanol. The solution was cooled and 6.96 g of 
NajS^O^ were slowly added. Two hundred milliliters of ethyl ether were 
added and the mixture stirred under nitrogen for one hour. The ether layer 
was separated, dried and reduced in volume. The oil was dried in vacuo : 
iH NMR (CDCI3) 6 6.9 (1 H, m), 5.6 (1 H, m) , 4.7 (1 H, m), 4.0 (2 H, t) , 
3.0 (2 H, m), 2.9 (3 H, s), 0.9 (3 H, t); UV 358 nm x^^^; Elemental analy- 
sis calculated %: C, 70.69; H, 9.97; N, 5.50. Found %: C, 70.76; H, 9.68; 
N, 5.88 (Ci5H25N02- ^H^O). 

1 ,4-Dihydro-l -methyl -3-pyridinecarboxylic Acid Decyl Ester/Decyl 1.4-Dihy- 
dro-N-methylmcotinate (27 ) 

A solution of 4.6 g of NaHC03 and 4.46 g of (19) (0.016 moles) was 
prepared in 5% aqueous methanol. The solution was cooled and 6.96 g of 
Na S were slowly added. To this solution was added 200 ml of ethyl 

2 2 1+ 

ether and the mixture stirred under for one hour. The ether layer was 
separated, dried and reduced in volume. The dried oil was orange in color: 
iH NMR (CDCI3) 6 6.9 (1 H, m) , 5.6 (1 H, m), 4.7 (1 H, m) , 4.0 (2 H, t) , 
3.0 (2 H, m), 2.9 (3 H, s), 0.9 (3 H, t); UV 359 nm \^^^\ Elemental analy- 
sis calculated %: C, 73.12; H, 10.39; N, 5.02. Found %: C, 73.16; H, 10.48; 
N, 5.03 (C^^H^gNO^). 

1 .4-Dihydro-l -methyl -3-pyridinecarboxylic Acid e-Phenyl ethyl Ester/g-Phen- 
ethyl 1 ,4-Dihydro-N-methylnicotiiriate (28) 

Four and six-tenths grams of NaHC03 and 3.89 g of (20) (0.016 moles) 
was dissolved in 100 ml of water. The solution was cooled and 6.96 g of 



59 

Na2S20i^ were added. Two hundred milliliters of ethyl ether were added and 

the mixture stirred over N2 for one hour. The ether layer was separated, 

dried and reduced in volume. The dried oil was orange in color: NMR 

(CDCI3) 6 7.2 (5 H, s), 6.9 (1 H, m), 5.7 (1 H, m) , 4.7 (1 H, m) , 4.2 (2 H, 

t), 3.0 (2 H, m), 2.9 (2 H, t), 2.8 (3 H, s); UV 355 nm x^^^; Elemental 

analysis calculated %: C, 70.18; H, 6.63; N, 5.46. Found %: C, 70.27; H, 

7.00; N, 5.10 (Ci5H^7N02- ■^H20) . 

Characterization of Dihydroberberine 

Distribution Coefficients 

Fifty milliliters of a cold 1 x 10"^ M solution of berberine (1) in 
pH 7.4 buffer were partitioned against 50 ml of CHCl 3 or 50 ml of 1-octanol. 
The concentration of (1) was determined spectrophotometrically in the organ- 
ic and aqueous layer. A stock solution of 2.7 x 10"3 M dihydroberberine 
hydrochloride (3) was made in methanol. An aliquot of this, sufficient to 
produce a 1 x lO"*^ M solution, was pipetted into 50 ml of cold pH 7.4 buf- 
fer and extracted immediately with either CHCl 3 or 1-octanol. After allow- 
ing for oxidation, the concentration of (1) in the organic and aqueous layer 
was determined spectrophotometrically. 
Potentiometric pKa Determination of Dihydroberberine (2) 

Due to the extreme water insolubility of (2) (< 3 yg/ml ) , all deter- 
minations were done in 25% methanol ic solutions. A titration curve was 
generated by adding 10 yl aliquots of NaOH to a 1 .0 mM solution of (3). 
The pKg was determined by inspection of the titration curve. During the 
experiments, all solutions were covered with a stream of nitrogen. 
Spectrophotometric pKa Determination of Dihydroberberine (2) 

The pKa of (2) was determined by measuring the absorbance difference 
at 355 nm in basic, acidic, and buffered media. The relationship that 
allows this determination appears below: 



60 



pKg = pH - log 



^obs ' '^HA 



a 



A" 



- a 



obs 



where a 



obs 



is the absorbance in buffer, a^- is the absorbance in base and 



HA 



is the absorbance in acidic media. 



Oxidation of Dihydroberberine (2) by Silver Nitrate 

Two hundred milligrams of (2) were dissolved in 95% aqueous ethanol . 
Upon addition of a 10% solution of silver nitrate, a black precipitate 
formed. Centrifugation and analysis of the supernatant shows stoichio- 
metric oxidation of (2). 

Oxidation of Dihydroberberine (2) by Diphenylpicrylhydrazyl Free Radical (DPP-) 

Two hundred milligrams of (2) were dissolved in acetonitrile. To this 
was added a solution of DPP- in acetonitrile which caused an immediate dis- 
appearance of the purple color due to DPP-. Ultraviolet analysis confirmed 
this oxidation. 

Oxidation of Dihydroberberine (2) by Concentrated Hydrogen Peroxide 

Two hundred milligrams of (2) were dissolved in 95% aqueous ethanol. 
A 30% solution of hydrogen peroxide was added and the system monitored by 
UV. The analysis demonstrated a rapid and complete oxidation of (2). 
Oxidation of Dihydroberberine (2) in Buffers 

The oxidation of (2) was determined by UV and an HPLC method. In the 
UV method, a solution of (3) was prepared and pipetted into buffers of 
various pH and at various temperatures. The changes of absorption at 
460 nm were measured with time. Data acquisition was facilitated by an 
Apple II microprocessor and an enzyme kinetic software package. In the 
HPLC method, two buffers, pH 5.8 and pH 7.4, were used. The samples were 
maintained at 37°C in a water bath. At certain times, 5 yl of the solu- 
lution were injected onto a uBondapak C,„ reverse-phase column, and the 



61 



1 



peak heights analyzed. The mobile phase was 60:40 acetonitrile : pH 6.2 
phosphate buffer and the flow rate was 2 ml/min. 

Quantitation of the Oxidation of Dihydroberberine (2) and Various Dihydro- 
pyridines (21) and (22) in Hydrogen Peroxide" 

Solutions of (3), (21) and (22) were prepared. An aliquot of these 
solutions was added to a standardized solution of hydrogen peroxide (H2O2) 
(0.18 M). The appearance of the 460 nm peak of (1) or the disappearance of 
the 359 nm peak of the dihydronicotinamides was measured. This determina- 
tion was made using the enzyme kinetics software package. 

Quantitation of the Oxidation of Dihydroberberine (21) and Various Dihydro- 
pyridines (22)-(28) in Plasma 

Freshly drawn 80% human plasma was obtained from Civitan Regional Blood 
Center. The oxidation of (3) was determined by HPLC and the oxidation of 
(3), (22), (23), (24), (25), (26), (27), and (28) by UV. In the HPLC analy- 
sis, a solution of (3) was added to 80% plasma and maintained at 37°C. At 
certain times, 1.0 ml of plasma was removed and treated with 3 ml of aceto- 
nitrile. The solution was centrifuged and 5 yl of the supernatant was 
analyzed by a yBondapak Cie reverse-phase column with a mobile phase of 
60:40 acetonitrile: pH 6.2 phosphate buffer. The peak heights were ana- 
lyzed and concentrations obtained from a standard curve. In the UV method, 
40% plasma was maintained at 37°C in a kinetic cell. A solution of either 
(3) or one of the various dihydronicoti nates was added to this and the 
appearance of the 460 nm absorbance of (1) or disappearance of the 359 nm 
absorbance of dihydronicoti nates was observed. 

Quantitation of the Oxidation of Dihydroberberine (2) and Various Dihydro- 
pyridines (22)-(28) in Liver Homogenate 

The determination of the rate of oxidation of (3) in a liver homogenate 
by an HPLC method and (3), (22), (23), (24), (25), (26), (27), and (28) 
by a UV method was performed. In the HPLC method, 14 g of fresh rat liver 



62 

were homogenized in 45 ml of cold phosphate-buffered saline. To this was 
added a solution of (3), and the system was maintained at 37.0°C. At vari- 
ous times, 1.0 ml of the solution was removed and the protein precipitated 
with 3 ml of acetonitrile. The sample was centrifuged and 5 yl of the 
supernatant analyzed using a yBondapak C^g reverse-phase column with a 
mobile phase of 60:40 acetonitrile: pH 6.2 phosphate buffer. The peak 
heights were analyzed and concentrations obtained from a standard curve. 
The UV method involved homogenizing 7 g of rat liver in 15 ml of pH 7.4 
phosphate buffer and diluting the homogenate to 200 ml (3.5% w/v). The 
homogenate was centrifuged and the supernatant was used. To this was added 
a solution of (3) or one of the dihydropyri dines. The appearance of the 
460 nm peak of berberine or the disappearance of the 359 nm peak of the 
dihydronicotinate was then measured. 

Quantitation of the Oxidation of Dihydroberberine (2) and Various Dihydro- 
pyridines (22)-(28) in Brain Homogenate^ 

Again, both an HPLC and UV method were employed in the determination 
of the rate of oxidation of (3), (22), (23), (24), (25), (26), (27) and 
(28). In the HPLC method, a solution of (3) was added to a 20% brain homo- 
genate in pH 7.4 phosphate buffer. The homogenate was maintained at 37°C 
in a water bath. At various times, 1.0 ml of the homogenate was mixed 
with 3 ml of acetonitrile. The sample was centrifuged and the supernatant 
analyzed by the same method used in the liver homogenates. In the UV 
method, 2 g of freshly obtained rat brain were homogenized in 33 ml of 
pH 7.4 phosphate buffer, yielding a 6.0% w/v homogenate. The homogenate 
was centrifuged and the supernatant was used in the determinations. To 
this was added a solution of (3) or one of the dihydronicoti nates. The 
appearance of 460 nm absorption of (1) or disappearance of the 359 nm peak 
of the dihydronicotinate was measured. 



63 

In litm Distribution of Berberine (1) and Dihydroberberine (2) in Whole 
Blood 

Solutions of (1) or (3) were added to 60 or 75 ml of freshly drawn 
heparinized sheep's blood maintained at 37°C. At various times, 4 ml of 
blood were withdrawn. The blood was centrifuged and the plasma removed. 
One milliliter of the plasma was treated with 9 ml of acetonitrile and the 
supernatant was analyzed spectrophotometrically. The entire volume of the 
packed red blood cells was treated with 8 ml of acetonitrile and centri- 
fuged. The supernatant was again analyzed spectrophotometrically. A stan- 
dard curve was obtained by preparing solutions of known concentration in 
plasma or packed red blood cells. Recovery from the red blood cells was 
71.4%. 

Effect of Glucose on the Distribution of Berberine (1) in Whole Blood 
Glucose was added to a volume of blood so that a concentration of 
200 mg% was obtained. The above procedure was then repeated. 

Animal Studies 

In Vivo Characterization of Berberine (1) and Dihydroberberine (2) 

White Sprague-Dawley rats, who weighed between 200-250 g, were anes- 
thetized intramuscularly with Inovar'^ (0.13 ml /Kg). Injections were made 
intravenously into the external jugular vein. The doses used include 
55 mg/Kg of (2) in dimethyl sulfoxide (DMSO), 55 mg/Kg of (3) in 20-25% 
aqueous ethanol , 55 mg/Kg of (1) in DMSO or 35 mg/Kg of (1) in DMSO. 
At certain times after the injection, the chest cavities of the rats were 
opened, the vena cava severed and the heart perfused with normal saline. 
Afterwards, the animals were decapitated and the brains removed. In cer- 
tain experiments, the lungs, liver, and kidneys were also excised. The 
organs were then homogenized in a minimal amount of water, usually 2 ml, 
and extracted with 8 ml of acetonitrile. A standard curve of (1) in the 



64 

organ homogenate was constructed. Analysis was performed by injecting 
5 yl of the supernatant onto a yBondapak Cis reverse-phase column with a 
mobile phase of 60:40 acetonitrile: phosphate buffer. The flow was 2 ml/ 
min. Under these conditions, the retention time of (1) was 3.8 min and 

(2) , 9.3 min. 

Slow Infusion of Dihydroberberine Hydrochloride (3) 

Rats were anesthetized and prepared as above. A dose of 55 mg/Kg of 

(3) was prepared in a volume of 1.0 ml. The vehicle was 20% aqueous etha- 

nol. This dose was infused into the external jugular vein over a period 

of either thirty or forty-five minutes. At the end of the perfusion, the 

animals were decapitated, their organs collected and analyzed. 

Effect of 1-Methyl-l ,4-dihydronicotinamide (21) on the Efflux of Berberine 
from the Brain 

Rats were anesthetized and prepared as above. Animals were injected 
with 200 mg/Kg of (21) in aqueous ethanol intravenously. After fifteen 
minutes, the standard dose of 55 mg/Kg of (3) was given. The animals were 
sacrificed at various times after the injection, selected organs were re- 
moved and homogenized, and the samples analyzed by HPLC. 
In lim Characterization of 1 -Benzyl -1 ,4-dihydronicotinamide (22) 

Rats were anesthetized and cut down as above. Doses between 60 mg/Kg 
and 400 mg/Kg of (22) were administered. At various times after the in- 
jection, the animals were perfused, decapitated, and the brains removed. 
The brains were then frozen in liquid nitrogen and stored at 0°C until 
they were analyzed, at which time the brains were thawed, homogenized 
in 2 ml of water, and extracted with 8 ml of acetonitrile. 

Analysis was made by HPLC using a uBondapak C^g reverse-phase column 
and a mobile phase of 40:60 acetonitrile: 1 x lO'^ M sodium heptanesul- 
fonate. The dihydronicotinamide had a retention time of 3.4 min and the 



65 

quaternary compound had one of 10,6 min at a flow rate of 2 ml /mi n. A 
standard curve was constructed in brain homogenates. 
Intracerebral Ventricular (icv) Administration 

Sprague-Dawley rats were anesthetized with 70 yl/lOO g of pentobar- 
bital sodium, and atropine sulfate (0.05 mg/Kg) was given, if necessary. 
Injections of (1), (6), and ^H-inulin (29) were made into the lateral 
ventricles of rat brains. The injections were made with the aid of a 
stereotaxic instrument and the site of the injection was -0.4 mm anterior- 
posterior, 1.5 mm medial -lateral and -3.0 mm dorsal -ventral relative to 
the bregma. The dose of (1) infused was 50 ug and the infusion volume 
was between 3 and 5 yl . The vehicle was DMSO and the infusion rate was 
5 yl/5 min. In several experiments, (1) was coinjected with 1000 yg of 
(6). In another set of experiments, a dose of 2.3 yCi of (29) was injected 
icv. 

At specific times after the infusions, the animals were decapitated. 
The brain was homogenized in 1.0 ml of water and (1) extracted with 4 ml 
of acetonitrile. Analysis of (1) was by HPLC with a mobile phase consist- 
ing of 50:50 acetonitrile: pH 6.2 phosphate buffer. A yBondapak Cig re- 
verse-phase column was used and a standard curve constructed using brain 
homogenates. For radionuclide analysis, the brains were homogenized in 
8 ml of water. Twenty-five hundredths of a milliliter of this homogenate 
were added to 0.75 ml of Soluene'^. After the sample dissolved, 12 ml of 
the scintillation cocktail were added. The samples were counted for five 
minutes and disintegrations per minute (dpm) were obtained by using a 
standard quench curve. 
Limited Metabolic Studies 

Rats treated intravenously (iv) with 55 mg/Kg of (1) or 55 mg/Kg of 
(3) were housed in metabolic cages. Urine was collected and extracted with 



66 

CHCI3. The aqueous layer was then extracted with 3-methyl-l-butanol . 
The organic layers were reduced to dryness and then reconstituted with 
a small volume (50 yl ) of methanol. This residue was used for HPLC and 
TLC analysis. Five microliters of each sample were analyzed by HPLC. 
A pBondapak Cis reverse-phase column and a mobile phase of 50:50 aceto- 
nitrile: pH 6.2 phosphate buffer were used. The TLC analysis consisted 
of spotting 5 yl of each sample on an alumina plate and eluting the sys- 
tem with cyclohexane: chloroform: acetic acid 45:45:10 or methanol. The 
plates were developed with iodine vapor or iodoplatinate spray reagent. 
Toxi ci ty 

White CD-I mice were employed in this study, the average mass of 
which was 22.6 ± 2 g. The mice were segregated into groups of 10, and 
8-10 groups were used in each study. The doses given were determined by 
preliminary studies in which the LDo and the LDioo were obtained using 
small groups of animals. The doses were then prepared in equal incre- 
ments between the two extremes, but there was additional emphasis placed 
at the lower end of the curve. Since this study was concerned with acute 
toxicity, the animals were injected intraperitoneal ly, and the number dead 
recorded after twenty- four hours. The groups were, however, observed an 
additional forty-eight hours to ensure an accurate appraisal of acute 
toxicity. Food and water were given ad libitum . The injection volume was 
75-100 yl. The data were analyzed by fitting them to a sigmoid curve and 
by the method of Probits. 

Anticancer Activity 

Male BDF mice (20.6 ± 0.3 g) were used in this study. A suspension 

of P388 lymphocytic leukemia cells was injected intraperi toneally (ip) 

(1 X 10^ cells) or intracerebral ly (2.5 x 10^ cells) . The survival time 

of animals treated with various doses of (1) or (3) compared to the controls 



.1 ■ 

67 

was recorded. Berberine or dihydroberberine hydrochloride were given ip 
3 times a day on day 2, 6, and 10 in 0.5% carboxymethyl cellulose. 



CHAPTER 3 
RESULTS AND DISCUSSION 
Synthesis and Characterization of Dihydroberberine 

The initial step in the application of the proposed drug delivery 
system to berberine (1) is the preparation of its dihydro adduct. The 
first synthesis of dihydroberberine (2) involved disproportionation of 
(1) in strong base and was performed by Gadamer in 1905. ^^'''^^^ The 
mechanism of this reaction involves nucleophilic attack of hydroxide to 
the carbon adjacent to the nitrogen resulting in the formation of a tran- 
sient amino alcohol. This intermediate collapses to oxyberberine and di- 
hydroberberine, presumably via a hydride transfer. Several other syntheses 
for (2) have appeared in the literature and these involve the direct re- 
duction of (1) by zinc amalgam, ,150 complex metal hydrides^^^ '^^^ or 
sodium borohydride. ^^3,i6if Historically, (2) has been of interest because 
of its spectroscopic properties, and as an intermediate in certain 
synthetic schemes. i^** The use of (2) as a drug or in a drug delivery 
system is novel to this thesis. 

In the present work (1) was reduced, as shown in Figure 3-1, by sodi- 
um borohydride in dry pyridine. Spectroscopic analysis showed that the 
yellow crystalline material produced was (2). The UV, IR, and MS are shown 
in Figures 3-2, 3-3, and 3-4 respectively, and are consistent with the 
assigned structure. ^^'7" The ^H NMR is presented in Figure 3-5, and the 
proton assignments in Table 3-1. The nmr is shown in Figure 3-6, and 
the corresponding carbon assignments in Table 3-2. These assignments were 
made by comparing (2) to a number of model systems. ^''^ The synthesized 




1 



70 



33 




200 300 400 500 600 

Figure 3-2. Ultraviolet Spectrum of Dihydroberberine (2) in 95% 
Ethanol 



71 




72 



o 



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ro 



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73 




74 




76 



Table 3-2. Carbon Assignments of the nMR of Dihydrobertjerine (2) 




Carbon Carbon 

Number PPM (g) Number PPM (6) 

1 150.301 11 111.457 

2 147.182 12 107.753 

3 146.597 13 103.659 

4 144.452 14 100.881 

5 141.528 15 96.202 

6 128.661 16 60.576 

7 128.466 17 55.848 

8 124.470 18 49.270 

9 122.033 19 48.929 

10 118.670 20 29.725 



(CDCI3: 78.267, 77.040, 75.732) 



77 

material gave an elemental analysis in good agreement with that predicted 
for (2). Also, the reduced material reacted rapidly with such oxidizing 
agents as hydrogen peroxide, silver nitrate, and 1 ,l-diphenyl-2-picryl- 
hydrazyl free radical (DPP-) to yield (1). These data support the suc- 
cessful synthesis of (2). 

The hydrochloride of dihydroberberine (3) was synthesized, as illus- 
trated in Figure 3-1, by treating a concentrated solution of (2), in methy- 
lene chloride with dry hydrogen chloride (HCl) gas. The hydrochloride 
reverts to (2) at pH above 7.0. Analysis of the regenerated material 
demonstrated no addition of HCl or any other nucleophile to the molecule. 
This addition is known to occur with several dihydropyri dines. The hydro- 
chloride is many times more soluble in aqueous solutions than the free 
base. 

If (2) is to be successful in a drug delivery system described in 
Figure 1-3, it should demonstrate a greater lipophilicity than (1). Di- 
hydroberberine is expected to be less polar than (1) because of the loss 
of the positive charge. To investigate the relative lipid solubility of 
(2) compared to (1), the two compounds were extracted with organic sol- 
vents and their distribution (partition) coefficients compared. 

The two solvent systems which were used included an octanol-pH 7.4 

phosphate buffer and a chloroform-pH 7.4 phosphate buffer. The alcohol - 

buffer system was chosen as one of the extracting systems because of its 

ability to, in some ways, mimic the partitioning of compounds in vivo . 

The data from Table 3-2 show that in both systems (2) has a high affinity 

for the organic phase while (1) exhibits a high affinity for the aqueous 
phase. 

The chemistry of dihydroberberine is largely a result of its enamine 
character. 1 Dihydroberberine, like all enamines, is in equilibrium with 



78 



Table 3-3. Distribution Coefficients for Berberine (1) and Dihydrober- 
berine Hydrochloride (3) in Chloroform/pH 7,4 Buffer and in 
1-Octanol/pH 7.4 Buffer 

Distribution Coefficient 
Compound Chloroform/pH 7.4 Buffer 1-Octanol/pH 7.4 Buffer 



Berberine (1) < 0.001 0.062 



Dihydroberberine 5.33 2.59 

Hydrochloride (3) 



79 

its corresponding imine and this unusual situation allows enamines, dihy- 
droberberine included, to be substrates in both nucleophilic and electro- 
philic reactions. This equilibrium tends to concentrate a negative charge 
on the carbon g to the nitrogen. Protonation usually occurs, for this 
reason, at the 3-carbon rather than the nitrogen. At physiological pH, 
a portion of the dihydroberberine molecules will exist in a C-protonated 
state. The pK^ which is an indication of the degree of this ionization 
is important since only the unprotonated free base is available for dif- 
fusion across membranes. The pKa of (2) was determined to be 6.80 ± 0.05 
by both a spectrophotometric and potentiometric method. At a physiological 
pH of 7.4, a substantial portion of (2) will thus exist in the unionized, 
freely diffusable form. Because of the water insolubility of the dihydro- 
berberine free base, all pKa determinations were carried out in 25% metha- 
nol ic solutions. 
Demethylation of Berberine 

A number of other synthetic schemes were explored in an attempt to 
obtain a method for preparing radiolabeled (1), should spectroscopic method 
prove too insensitive. A radio! abelled compound would also greatly expe- 
dite whole body distribution studies. In radiolabeling a compound, one 
of the important factors governing the selection of a synthetic route is 
yield. The scheme which was chosen involves pyrolyzing (1) at 200°C. ^ ''^ ,i7i 
Berberine loses methyl chloride, as shown in Figure 3-7, to form the deep 
purple zwitterionic berberrubin. This method would allow the placement 
of either a ^'♦C or label at the nine position of (1) by reacting ber- 
berrubin with the appropriately tagged methyl iodide or methyl sulfate. 
Methyl ati on of berberine with cold methyl iodide was performed to demon- 
strate the viability of the scheme. 



80 




81 



Theoretical Studies on the 
Dihydropyridine Pyridinium Redox System 

If the drug delivery system described in Figure 1-3 is to be used to 
its potential, then, a knowledge of its basic chemistry is required. The 
reason for this is not only to understand the subtle chemistry inherent 
in this particular system, but also to allow prediction, extrapolation 
and generalization of the system to other examples. The ability to dis- 
cern, for instance, those factors that add to or detract from the stability 
of a molecule would allow attenuation of a particular property by mole- 
cular manipulation. A thorough chemical knowledge of a particular com- 
pound would also allow an intelligent prediction as to its suitability 
to the scheme. 

While a few ionization potentials have been measured, the general in- 
stability of dihydropyri dines often precludes their investigation by ex- 
perimental means. Because of this limitation, these compounds have lent 
themselves well to theoretical study. While the dihydronicotinamides, 
because of their biological relevance, have been the subject of copious 
reports, larger di hydro systems have received little attention.^'^^'^'^^ 
In order to gain a greater chemical insight into the proposed drug deliv- 
ery system in general, and the berberine (1) t dihydroberberine (2) sys- 
tem in particular, a theoretical investigation was undertaken. 

Berberine is a rather large molecule and, as such, would be expected 
to present problems in terms of computational time. It must be remembered 
that 3N-6 (where N is the number of atoms) independent variables are re- 
quired for a molecule and large molecules can easily cost $8000-$10000 
in computer time. Because of this, and also in an attempt to generalize 
the calculations so that they would be applicable to a number of compounds, 
a model was developed for the (1) t (2) system. The compounds chosen as 



82 

the model, 3H,4H-dihydro-7,8-dihydroxybenzo[b]quinol izinium ion (30) and 
3H,4H,6H-trihydro-7,8-dihydroxybenzo[b]quinolizine (31) along with their 
numbering protocol, are shown in Figure 3-8. 

This quaternary and its dihydro adduct, while simpler than the 
(1) (2) system, do not differ greatly in structure or in general chem- 
istry. Compounds (30) and (31) have not been investigated and no mention 
of them has appeared in the literature. Some theoretical work on the 
unsubstituted benzo[b]quinol izinium ion and much on the isoquinoline mole- 
cule has been published. The benzo[b]quinolizinium ion was investigated 
by Galasso in 1968 and charge densities and Ti-bond orders were reported. ^'^'^ 
All of these reported studies have employed simplistic theoretical treat- 
ments such as PPP,i78 iOC-a)-technique,i79 hM0,180 sSP.i^i and CNDO/2.182 

The corresponding dihydrocompounds have not been studied. 

In order to investigate the isoquinoline model (30) t dihydroisoqui no- 
line model (31) system, a MINDO/3 method was selected. This approach 
is a semi-empi rical self-consistent field molecular orbital method in which 
all valence electrons are treated. The MINDO/3 program is the culmination 
of the series MINDO,!^'^ MIND0/2,i85»i86 g^d MIND0/2'.i87 jhis program was 
developed to solve chemical problems quickly and efficiently using a quan- 
tum mechanical framework. Unlike the methods of Pople, whose major aim 
was to reproduce nonempi rical calculations, M.J.S. Dewar parametrized 
MINDO so that the system would produce useful and chemically accurate data. 
This program is extremely versatile and has been applied to a number of 
chemical problems including reactions. Only the most cursory details of 
MINDO/3 are appropriate here, as the subject is well reviewed elsewhere. 

In quantum mechanical approaches like MINDO, a molecular orbital is 
the result of the interaction of a wavefunction of an electron with nuclei 
and other electrons present in the molecule. Mathematically, the 



83 



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84 

Hamiltonian for such a system consists of the kinetic energy term for the 
movement of the electrons and the potential energy terms for electron- 
nuclei attraction and electron-electron repulsion. 

In systems with only one electron, e.g. H, H2'*" or He"*", the differen- 
tial equation which constitutes the Hamiltonian can be separated and 
exactly solved. If, however, more than one electron is present, the elec- 
trons interact and the differential equation is no longer separable or 
exactly solvable. Because of this problem, approximations and simplifi- 
cations are incorporated into the Schrodinger equation. These include 
considering the molecular orbital {"v) as a linear combination of atomic 
orbitals (<{.): 

>f = I C. 4.. 

Additional simplifications are obtained by neglecting certain electron 
repulsion terms since these values are close to zero. These approximations 
allow the application of quantum mechanical approaches like MINDO to rather 
complex chemical problems without extremely large expenditures of capital 
or computer time. 

The MINDO/3 program provides the following data: optimized geometries 
of the most stable ground state conformation at 25°C, the heat of forma- 
tion (AHf) in kcal/mole at 25°C of the optimized structures, the atomic 
charge distribution, the dipole moments, the total electronic energy, the 
vertical ionization potentials, the bond order matrix, and the eigen vec- 
tors and eigen values. 

The eigen values and eigen vectors allow a thorough examination of 
the contributions of the individual atomic orbitals to the molecular or- 
bital and thus of the electronic structure of the molecule. This can be 
indicative of many of the chemical proclivities of a molecule. The MINDO 



85 



approach allows one to examine compounds and reactions which are not 
approachable by conventional means. For example, the stability of very 
reactive species can be assayed, their structures determined, and quali- 
tative aspects of their chemistry elucidated. 

The input for the program includes, for each atom, the atomic number, 
the approximate bond distances between adjoining atoms, the approximate 
bond angle, and the approximate twist angle out of the plane. As previously 
mentioned, the number of parameters needed is 3N-6 where N is the number 
of atoms. Convergence is assumed when the difference between two succes- 
sive calculations is less than 0.1 kcal/mole. 

The two models (30) and (31) were analyzed and the results are pres- 
ented in Table 3-4 through 3-8. The heat of formation calculated for (30) 
was 95.1 kcal/mole and that of (31) was -39.3 kcal/mole. While the use of 
AHf for comparisons is restricted to structural isomers, the relative sta- 
bility of a system can be calculated by comparing the differences in AHf 
(AAHf) from one system to another. The AAHf of the isoquinoline model 
(30) t dihydroisoquinoline model (31) pair is 134.4 kcal/mole. As shown 
in Table 3-9, this value is smaller than that obtained from simple dihy- 
dropyridine t pyridinium systems indicating a greater stability of (31) 
relative to simple dihydropyridines . The stabilization of (31) indi- 
cates that it should be less reactive than simple dihydropyridines . The 
basis of this stabilization is derived from the extended aromatic conjuga- 
tion of (31). Another contribution to the relative stabilization can be 
seen by an examination of the highest occupied molecular orbital (HOMO) 
which is represented in Figure 3-9 as a linear combination of atomic orbi- 
tals. In looking at the HOMO, only the magnitude of the coefficients is 
important since the sign simply represents the phase of the orbital. A 



87 



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91 



Table 3-9. Differences in the Heats of Formations (AAHf) of (30) z (31), 
2-PAM t 1 ,4-Dihydro-2-PAM and 2-PAM t 1 ,6-Bihydro-2-PAM 



[AAHf I 



134.40 
kcal/mole 




2-PAM 1 ,6-dihydro-2-PAri 



"^HOMO ^ ^-"^"^^ Pz^^i) O-Q'^^ p^CCj - 0.221 p^lCg) - 0.247 p^(Cj 
+ 0.479 p^CCs) + 0.291 p^CCg) - 0.074 p^CCy) - 0.174 p^(C8) + 0.013 p^CCg) 
+ 0.018 p^CCjo) + 0.130 P^CCii) + 0.343 p^{Ci2) + 0-106 P2(Ci3) - 0.284 p^{C 
+ 0.005 sCHis) + 0.006 s{H^^) - 0.085 p^iO^j) - 0.079 p^COig) - 0.008 sCHig) 
+ 0.008 s(H2o) - 0.010 s(H2i) + 0.041 SCH22) - 0.034 s(H23) + 0.113 siH^^) 
- 0.082 s(H25) + 0.138 s(H26) - 0.179 s(H27) - 0.005 s(H2g) - 0.092 SCH29) 



Figure 3-9. The Highest Occupied Molecular Orbital of the Dihydroi 
line Model (31) 



93 

relatively large contribution to the HOMO is made by the methylene hydro- 
gens 27, 28 and 24, 25 and the nonaromatic carbon, C^. This phenomenon 
is termed hyperconjugation. Hyperconjugation also occurs in simple 1,2- 
and 1 ,4-dihydropyri dines but, because of the additional methylene inter- 
actions, the effect is slightly larger in the case of (31).i3° The large 
contribution to the HOMO by the nitrogen lone pair is also noted. 

The vertical ionization potentials (IP) of (30) and (31) were calcu- 
lated using Koopman's Theorem which simply states that the ionization po- 
tential is the negative of orbital energy. The calculated values are 
presented in Table 3-4 and are 11.56 eV for (30) and 7.19 eV for (31). 
In addition, the character of the orbital which loses the electron can be 
obtained by examination of the HOMO and, for (30), a u-type system is in- 
volved while for (31) a mixed -n-P^ type orbital occurs. These values are 
similar to values obtained from other systems and are consistentwith the 
molecular structures. The dipole moments calculated for (30) and (31) 
also appear in Table 3-4 and, again, are consistent with the molecular 
structure. 

It is instructive to compare changes that occur upon reduction of 
simple dihydropyri dines to those that occur upon reduction of (31). This 
cmparison demonstrates the similarity in chemistry between simple and 
more highly conjugated dihydropyri dines and also demonstrates the extreme 
usefulness of the computational method. Tables 3-10 to 3-12 are comparisons 
of the pyridine (32) :^ dihydropyri dine (33) system and the pyridinium 
nucleus of (30) :J (31) system. The values for the (32) :t (33) system 
were calculated by a MINDO procedure by Bodor and Pearlman in 1976.^30 j^g 
numbering of these various compounds appears in Figure 3-8. 

Table 3-10 shows the bond lengths in the isoquinoline model system, 
(30) :^ (31), and the simple pyridine system, (32) ^ (33). Upon reduction 



94 



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in 






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s- 
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00 


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CO 


CO 




00 






CO 


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s- 

>> 

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c 



CL 



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CD 




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to 

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r— 
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1 — 
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co 















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CO 















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I 

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CO I— 

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CO 



CO 

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cn 







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1 




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4-> 


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CD 




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c 


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95 

of (30), the bond connecting carbons 5-6 shortens, indicating a greater 
double bond character at this location, while the bonds between carbons 2-3 
and between the carbon and nitrogen at position 1-2 lengthen, indicating 
an increased single bond character at these locations. This correlates 
well with the structural formalism and also mirrors those changes that 
occur in the reduction of (32) to (33). A similar study of bond angles is 
presented in Table 3-11. 

The charge densities at specific atoms can be indicative of the type 
of chemistry that a compound undergoes. A study of the charge densities 
of atoms in (30) t (31) is presented in Table 3-7 and a comparison of this 
system to the (32) t (33) system appears in Table 3-12. The charge densi- 
ties of the pyridinium nucleus of (30) reveal the most highly charged de- 
ficient center is at carbon Cp. One would expect nucleophilic attack at 
this electropositive position and, in fact, this is what is observed. 
These observations can be extended to berberine (1) since it is known that 
hydroxide, hydride, and acetonide attack (1) at this position. In general, 
nucleophiles attack pyridines at the carbon adjacent to the nitrogen. 
The charge densities of the atoms in (31) indicate a highly electronegative 
center at Cg. One would therefore expect protonation and electrophilic 
attack at this location. This, again, is borne out experimentally. In 
the case of dihydroberberine (2), protonation as well as alkylation occurs 
here and, in general, this type of reaction is well known in the chemistry 
of enamines . ^^^"^^3 These properties and trends are also seen in the 
(32) t (33) pair. 

The planarity of a pharmacologically active aromatic molecule, espe- 
cially antineoplastic agents, is an extremely important parameter and many 
correlations between activity and toxicity and planarity have been made. 



96 



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98 

This characteristic is accessible by MINDO/3. The program calculates a 
dihedral angle which is a measure of the deviation from planarity of a 
molecule. Pyridine (32) is planar and 1 ,2-dihydropyridine (33) is planar 
within one degree. The dihedral angles of (30) J (31) are shown in 
Table 3-8. In the case of (30), the overall deviation from planarity is 
less than 3.6°. In berberine (1), which has an additional benzene ring 
annul ated at the C7-C3 position, this difference should be less because 
of the planarizing effects of the added aromatic system. This would tend 
to cast doubt on the proposal that the reason berberine does not inter- 
calate into deoxyribonucleic acid (DNA), as well as totally aromatic mole- 
cules such as coralyne, is due to the buckling of the C ring. ^25,155 
This nonplanarity is attributed to the partial saturation of that ring. 
The present study, however, indicates that this deviation is slight and 
probably plays a minor role in attenuation of the action of (1). 

A more plausible reason for the lower intercalative ability of (1) 
is because of lower electronic interactions. When a molecule intercalates 
into DNA, there exists an electronic interaction between the base pairs 
of DNA and the TT-cloud of the intercalating aromatic compound. The greater 
the stabilization of this complex, the greater is the DNA-molecular inter- 
action. In berberine, there is a partial destruction of the aromatic 
system which lowers any electronic interaction. The hydrogens added to 
the C-ring act to increase the effective thickness of the molecule and 
this may play a role in decreasing macromolecular complexation. These 
structural concerns are apparent in Figures 3-10 and 3-11, which are the 
fully optimized structure for (30). 

The dihydro model (31) contains one more sp^ center than does (30), 
and this has a slight depl anarizing effect with the molecule twisting 9.1° 



100 







QJ ^ 1 — 




^ -t-> D- 




+-> 








<+- ro ^ 




O ^ ■•-> 




+-> 




c o 




o o +-> 




•I— 10 




+J i- 








E OJ 1— 




i- +-> 13 




O C tJ 




H- O •!— 




C •!— "O 




O s- c 




O O CD 




c 




0) to i- 




1 — ■!— <D 




^ Q- 








4J CJ CO 




</l T- -1— 




> 




+-> CM 




10 10 










t— fO 




O) 




-CI I.O 




+-' • C\J 




o 




<+- o to 




o un S 




OvJ o 




Ol +-> 




c 4J <; 




• r— 




3 £= 




(O N (U 








Q n s 




4J 




-O QJ 




GJ 1— J3 




+J O) 




to T3 to 




•1- O •■- 




to SI X 




to c£ 








1 c o 


(U 


S_ -1- •!- 


Dl 


<U .— E 


03 


+J o o 


a. 


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0) 


E =5 S- 




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+-) 


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to C 


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I 

CO 



101 

out of the plane. In applying the same considerations to this molecule 
as to (30), one would expect that this system would interact less with 
DNA than would (30). This is due to the further destruction of the aro- 
matic nucleus resulting not only in a more nonplanar structure, but also 
in a structure with a lower propensity to interact electronically with 
DNA. The loss of the positive charge should also reduce macromolecular 
intercalation because of the lowered coulombic interaction between (31) 
and the anionic phosphate backbone of DNA.ise in extending these results 
to dihydroberberine (2), one would predict a lower cytotoxicity and, there- 
fore, toxicity of (2) relative to (1). The optimized structure of (31) 
at 25°C is presented in Figures 3-12 and 3-13. 

To summarize, the MINDO/3 calculation predicts the dihydro model (31) 
and, presumably, (2) to be more stable than simple dihydropyri dines because 
of extended conjugation and hyperconjugation. The model (30) is predicted 
to undergo nucleophilic attack at the carbon adjacent to the nitrogen, and 
protonation and electrophilic reaction at the carbon B to the nitrogen of 
the enamine. The calculations show localization of double and single 
bonds on the reduction of (30) to (31). These data are in good agreement 
with what is known about chemistry of this genre of compounds. The calcu- 
lation suggests that the reason berberine does not intercalate as well as 
totally aromatic compounds is not because of steric problems associated 
with the carbon skeleton but, rather, electronic differences and the steric 
effects of added hydrogens and, finally, MINDO/3 predicts that (31) and 
presumably, (2) are less toxic than (30) and (1). These results should 
be applicable not only to the (1) t (2) system, but other conjugated di- 
hydropyri dine systems as well. 



102 



1 




Figure 3-12, A Computer-assisted Drawing of the Most Stable 
Conformation of the 1 ,2-Dihydroisoquinol ine 
Model (31) at 25°C 



103 



I 

o 
s_ 

-a >, 
s: fo 

•r- C 

Q -r- 

I cn 

CM <T3 

" s 

I — I— I O) 

CD 

(U fZ (0 
J= fO D- 

+J QJ 

O ^ ■•-> 
-P 

C 4- 

O O O 
•I- in 

+-> o 

<B -o c 

E CD fO 

5- ■!-> r— 

O C Q- 

"4- O) 

C ■<- (U 

O S- -C 

O O +-> 

o) CO o 



+-» 



> 3 
U 

(/) 

•1- -a 

I— <u 

Q. 
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(U 
C- 



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o un 

CM 

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S- I— (O 

Q cn 

— 'CM 

T3 

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(/) "O o 
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0) c 
C O) 



I 

S- 



3 

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o 
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3 

CT CO 
O T- 
CT X 
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I 



104 

Further Studies on the Biological and 
Chemical Properties of Dihydroberberine 

The rate and nature of the oxidation of the dihydropyri dines included 
in the delivery scheme is important to the proper functioning of the system. 
A dihydropyri dine must be stable enough to be formulated and stored. The 
in vivo rate of oxidation must, however, be rapid enough to efficiently 
transform the delivering species and thereby avoid competing metabolisms. 

The rate of oxidation of dihydroberberine (2) was therefore studied 
in a variety of media, and in a number of different situations. One prob- 
lem which hampered these determinations was the extreme water insolubility 
of the free base. The rate of oxidation of (2) was determined by both 
HPLC and UV methods. The UV procedure involved measuring the appearance 
of the 460 nm absorbance of berberine (1) with time, while the HPLC deter- 
minations were made by calculating the appearance of the absorbance due to 
(1) or disappearance of the absorbance due to (2). In most cases agreement 
between the two methods was good. In all determinations the spectrum of (1) 
showed no change within the timeframe of the experiment. Initial oxida- 
tion studies were performed in areated buffer. At a pH of 7.4, (2) oxi- 
dized very rapidly and erratically. Buffers of lower pH were then used 
to partially stabilize (2) by shifting the equilibrium in favor of the 
hydrochloride in an effort to yield a more reproducible system. This 
shift reduces the electronic density at the nitrogen, and precludes the 
participation of the nitrogen lone pair in oxidative reactions. i'^^ jhg 
lower pH also greatly facilitates solubilization. The rate of oxidation 
of (2) at a pH of 5.8 is shown in Table 3-13, and the spectral changes 
that are characteristic of this oxidation are shown in Figure 3-14. 
Although the correlation coefficients are not good, second order kinetics 
are indicated with a calculated second-order rate constant of 44.4 ± 0.53 



105 



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c 
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+-> 

10 

+-> 

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c 
o 
o 



I c 

O) o 

S- •!- 

S- 4-> 
O ITJ 
CJ I— 



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c 

j_i CO 



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106 




c 

Z. c 

O) -r- 
-Q E 
S- 

QJ O 
CO I— 

O >, 
+-> S- 
O) 
c > 
O O) 

4-J CU 

« T3 
■O (B 

•-- e 

o <u 
i. 
c: CU 
o 3 

Cl 

3 (/) 
(U 
O 

CM to 
' S- 
I— 

<u 

c • 

•I- o 

S- o 
O) li) 
^ CM 

s- 

j3 res 
o 

s- s- 

■O (U 



C2 CO 

o +J 
CO 

<u c 

O) I/) 

c o 

rO ^ 

x: D- 

o 

CO 

(13 m 
s- 

4-) ni 

(J Q. 

a. c 

00 T- 



33Nvadosav 



2! 



107 

£./mole sec. A number of oxidants were investigated to study the oxida- 
tion of (2). Unfortunately, these produced rates of oxidation which 
were far too rapid to analyze by simple means. 

The rate of oxidation of (2) in various biological media was also 
determined and these results are presented in Table 3-13. Second-order 
kinetics were again observed. In brain homogenates the correlation co- 
efficients were much higher than those obtained in buffer. In the major- 
ity of the determinations, pseudo first-order rate constants at specific 
concentrations were calculated. The values obtained allow a comparison 
from system to system. The ti, for example, of oxidation of a 5 x 10"^ M 
solution of (2) in plasma, liver homogenate, and brain homogenate was 
calculated to be forty-eight minutes, thirty-one minutes, and approximately 
twenty-five minutes, respectively. The oxidation of (2) by a second-order 
process is very characteristic of many dihydropyri dines. 

The relative stabilities of dihydropyri dines were investigated by com- 
paring their rates of oxidation in dilute hydrogen peroxide. This system was 
reproducible and, as shown in Table 3-14, gave data of good quality. The 
ti calculated from the pseudo first-order rate constants obtained at 1 xlO"'*M 

2 

for (2), 1-methyl-l ,4-dihydronicotinamide (21), and 1-benzyl-l ,4-dihydro- 
nicotinamide (22) were 25.6 min, 1.2 min, and 11.5 min, respectively. 
These results are consistent with the greater stabilization of (2) compared 
to simpler systems, which was predicted by the theoretical calculations. 
The rate constants are relatively small because of the slightly acidic nature 
of the peroxide. At the end of the experiment, (1) was analyzed to make 

sure that nucleophilic addition of the peroxide to (2) did not occur. 
The Mechanism of Oxidation of Dihydroberberine 

A knowledge of the mechanism of oxidation of dihydropyri dine could 
be helpful in applying the drug delivery system. The mechanism of oxidation 



108 



Table 3-14. The Relative Rates of Oxidation of Dihydroberberine (2), 
1 -Methyl -1 ,4-dihydronicotinamide (21), and 1-Benzyl-l ,4- 
dihydronicotinaniide (22) in Dilute Hydrogen Peroxide 



Pseudo First 
Compound Order Rate Constant 

(2) 4.51 X lO"** 

(21) 9.83 X 10'3 

(22) 1.00 X 10"3 



Relative Rate 
Correlation of Oxidation 

0.998 1.0 

0.99999 21.7 

0.999 2.2 



109 

of simple dihydropyridines , particularly dihydronicotinamides , has been 
extensively studied since these partial structures occur in the NADHJNAD'*' 
system. Many models of this system have been used and most are simple, 
substituted 1 ,4-dihydronicotinamides . 

In the classic work of Abeles and Westheimer, the oxidation of sub- 
stituted 1 ,4-dihydronicotinamides by thiobenzophenones was studied. 1^*+ 
Like most dihydropyridines, these exhibit second-order kinetics: first- 
order with respect to the dihydropyridine and first-order with respect 
to the thiobenzophenone. The mechanism of oxidation proposed by this 
group was that of a concerted hydride transfer from the dihydronico tin- 
amide to the thiocarbonyl carbon. This mechanism has been modified over 
the years. Most recently, Ohno has described a system in which the 
oxidation proceeds through a charge transfer complex. ^^^'^^^ The initial 
step in this process is an electron transfer, followed by a proton trans- 
fer followed, in turn, by a subsequent electron transfer. In free radi- 
cal oxidations Eisner, using substituted 1 ,4-dihydronicotinamides and the 
oxidant, di phenyl pi crylhydrazyl free radical (DPP*), again found a second- 
order oxidative process with the rate-determining step being the initial 
abstraction of a hydrogen. This is followed mechanistically by the 
formation of the quaternary compound. The oxidation of other dihydropy- 
ridines, such as the free radical oxidation of dihydroanthracene or dihy- 
drophenanthrene, has also been reported. ^oi >202 ji^g kinetics of enzymatic 
oxidation of dihydronicotinamides have also been studied. In 1980 Porter 
and Bright published an article on the oxidation of substituted 1,4-dihy- 
dronicotinamides by lumi flavins and old yellow enzyme. 2° ^ Kinetically, 
a second-order oxidation was found to take place, mediated by a charge 
transfer or bi radical complex. 



110 

The mechanism of oxidation of dihydropyri dines is, therefore, depen- 
dent on the oxidant and the conditions under which oxidation takes place. 
A series of experiments was performed to investigate the mechanism of oxi- 
dation of simple dihydronicoti nates and (2), and to determine if the oxi- 
dation of the dihydropyri dines is mediated by an enzyme or by some other 
species such as dissolved oxygen. 

In these experiments a homologous series of 1 -methyl -1 ,4-dihydronico- 
tinic acid esters was synthesized. The NMR of a representative compound 
is presented in Figure 3-15, and the corresponding proton assignments in 
Table 3-15. The rate of oxidation of (22), (23), (24), (25), (26), (27), 
and (28) in 40% human plasma, 6% brain homogenate, or 3.5% liver homogenate 
was measured. This was done by determining the rate of disappearance of 
the 359 nm absorption of the dihydronicotinamide with time. Since the 
rate of ester hydrolysis is slower for nicotinic acid esters and much 
slower for 1 -methyl nicotinic acid esters than the values obtained, the 
results clearly represent the oxidation process of the dihydropyri dine 
and not hydrolysis. In all determinations the concentration of the dihy- 
dropyri dines was 5 X 10" 5 M. The results of this experiment are shown 
in Figure 3-16 and Table 3-16. Both in plasma and in buffer, the rate 
of oxidation as measured by the pseudo first-order rate constant, is rela- 
tively slow and the correlation coefficients are relatively small. There 
is also little effect on the rate constants by the molecular structure. 
This indicates a nonspecific oxidative route. In organ homogenates, there 
is a marked acceleration in the rate of oxidation as well as an increase 
in the correlation coefficients. There is also a large dependence upon 
the rate by the structure of the molecule and, in general, as the chain 
length increases, the rate decreases. These three changes - acceleration 
of the rate, linearization of the data, and the greater reliance of the 



in 




112 



Table 3-15. Proton Assignments of the NMR of the 1 -Methyl -1 ,4-di hydro- 
nicotinic Acid Ester (27) 




OCH2(CH2)8CH3 



CH3 



Proton PPM (6) 

a 6.9 

b 5.6 

c 4.7 

d 4.0 

e 3.0 

f 2.9 

g 0.9 



113 



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m 




N 

OD 
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c 
o 



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o 
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s- 

0) 

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c c 

<C -r- 13 

+-) 

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+-> o 

O S- QJ 

a -o -M 

•I- >, n3 

c x: c 

o •!- (U 

S- Q CD 

T3 I O 

>,-^ E 

J= " o 

•1- 1— ni 

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1 r— c 
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.— C S- 

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I— CQ 
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O) "O •> 

c « 



(/I CO 
3 CM 
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S- « ra 

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+-> 

fO 

o c 

oi 

CD 

c o 
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o 

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115 

rate on structure - indicate the involvement of an enzyme in the oxida- 
tion. One of the enzymes which is said to be responsible for the oxida- 
tion of dihydropyri dines is NADH dehydrogenase. ^o'* Since this family of 
enzymes is membrane bound, it would be present in the organ homogenate 
but not in plasma. The results are consistent with this distribution. 
Enzymes involved in the oxidation of dihydropyri dines have as their endo- 
genous substrate NADH, This molecule is not substituted at the amide 
nitrogen and one would expect compounds which more closely resemble NADH 
to be better substrates for the enzyme than molecules which greatly devi- 
ate from this structure. One would predict that the shorter chain ana- 
logs (23) and (24), and the N-benzyl compound (22) would be oxidized 
more rapidly than the longer chain analogs and this is, in fact, the case. 
An additional observation which supports this hypothesis is that as the 
homogenates age, the rate constants as well as the correlation coeffi- 
cient decreases. This is consistent with the time-dependent denaturation 
characteristic of this type of enzymatic system. 

These trends also occur in the oxidation of (2). In plasma and buf- 
fer, the data indicates a relatively slow oxidation with poor correlation. 
In brain homogenate, there is an acceleration and a linearization in the 
rate constants. These results indicate that although oxidation of (2) 
can be mediated by oxygen, in tissues like the brain and liver, an enzy- 
matic oxidation can occur. The effect of protein binding on the rate of 
oxidation of (2) was investigated by changing the concentrations of the 
homogenates. One would expect (2) to bind to proteins but the effects 

of this complexation on oxidation were not large. 
Membrane Permeability of Dihydroberberine and Berberine 

In order to investigate the relative ability of dihydroberberine (2) 

to penetrate membranes, the behavior of (2) and (1) in a model system was 



116 



observed. The model system which was chosen in this study was that of 
the red blood cell. In this experiment (1) or (2) were placed in a known 
volume of whole blood, and at various times the concentration was deter- 
mined in the plasma or packed red blood cells. The results are shown in 
Figure 3-17. As one can see, the initial rate of penetration of (2) into 
red blood cells is rapid and greater than that of (1). The initial con- 
centration is also higher. This affinity for the red blood cells is mir- 
rored by a disappearance of (2) from the plasma. With time, equilibra- 
tion occurs in the system. Berberine penetrates the red blood cell slow- 
ly and reaches a maximum concentration much later than does (2). This is 
another indication of the increased membrane mobility of (2) relative to 
(1). The two curves finally converge to the same value, as the oxidation 
of (2) to berberine takes place. Creasey indicated that there was a re- 
lationship between glucose transport and the transport of berberine into 
red blood cells. i'*^ jq investigate this possibility, the behavior of ber- 
berine in a red blood cell system which contained 200 mg% glucose was ob- 
served. As shown in Table 3-17, there is very little effect of glucose 
on the entry of berberine into the red blood cells from the plasma. 

In Vivo Studies 

The preliminary studies indicate that dihydroberberine possesses all 
of those characteristics required of a compound which is to be applied to 
the drug delivery system proposed in Figure 1-3. The substantiation of 
this drug delivery scheme requires not only the demonstration of the deliv- 
ery of (1) after administration of (2) but also the specific retention of 
(1) in the brain. The first priority of the in vivo system was to show 
delivery into the brain of (1). The protocol used in these studies in- 
volved injecting rats with either berberine (1), dihydroberberine (2), 
or its hydrochloride (3). After a period of time the chest cavities of 



117 






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118 



Table 3-17. The Effect of Glucose on the Movement of Berberine into 
Red Blood Cells 





Berberine Concentration (uQ/ml) Plasma^ 


Time (min) 


Control 


+ 200 mg% Glucose 


c 


360.94 


352.29 


30 


300.39 


309.04 


60 


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276.48 


90 


267.29 


255.62 


120 


259.17 


272.92 


^This is a 


representative experiment selected from a 


group of three 



119 

the animals were opened, the heart perfused with saline, and the brain 
removed. High pressure liquid chromatography was used in analysis. 

The initial experiments showed that after administration of 55 mg/Kg 
of (1) in dimethyl sulfoxide (DMSO), no (1) could be detected in the brain 
at any time. When, however, 55 mg/Kg of (2) in DMSO were administered, 
a high concentration of (1) was observed in the brain as shown in Figure 
3-18. The free base was not exceptionally stable in solution, however, 
and was also very water insoluble. For these reasons the hydrochloride 
(3) was prepared and used in subsequent experiments. If 55 mg/Kg of (3) 
in 20% aqueous ethanol are injected systemically, the concentration of (1) 
in the brain is again found to be relatively high. This is presented in 
Figure 3-19. The concentration of (1) achieved in the brain after admin- 
istration of (2) or (3) was similar (approximately 50 yg/g tissue). The 
loss of (1) from the brain after administration of (3) is slow and the 
t^ of this loss, calculated from the terminal portion of the log concen- 
tration versus time relation, is approximately eleven hours. 

It should be emphasized that in these experiments only the concen- 
tration of (1) was measured even though unoxidized (2) was present. To 
obtain a more complete picture of the behavior of (1) in the brain, the 
total berberine concentration, i.e. (1) and (2) was measured and this 
appears in Figure 3-20. The concentration of (2) is high at early time 
points but diminishes rapidly. The initial rate of disappearance of (2), 
obtained by subtracting the concentration of (1) in the brain from the 
total concentration (Figure 3-21), yields a t^ of thirty-four minutes 

2 

which is of the same magnitude as the value obtained from brain homogenates. 
The slope of the terminal portion of the curve in Figure 3-20 yields a 
ti of 5.7 hours. 

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This indicates a faster efflux for the total berberine concentration 
than for the efflux of (1) alone. The reason for this is that, aside 
from efflux of (1) from the brain, a component of the oxidation of (2) 
and for the efflux of (2) also occurs in this rate. Since the oxidation 
of (2) is not immediate, a significant quantity of it is present at later 
times and this population of molecules will redistribute out of the brain 
as a function of the blood concentration of (2) as would any lipophilic 
compound. This added equilibrium complicates the kinetics of the original 
scheme. 

The distribution of berberine (1) in various tissues after an intra- 
venous dose of 35 mg/Kg is shown in Figure 3-22. The lower dose was used 
because of the higher toxicity of (1). These results show a high concen- 
tration of (1) in the kidney and, to reiterate, no quanti tatable amount 
in the brain. Berberine is rapidly lost from the tissue and this contri- 
butes to its relatively short biological t^. The distribution of (1) in 
the tissues has been previously studied. ^05-209 jhg results obtained from 
a number of these studies are consistent with those reported here. Ber- 
berine is rapidly lost from the tissues and effectively excreted by the 
kidney and by a biliary route. In literature reports the concentration of 
(1) in the brain was always undetectable or the lowest of all other tissues 
examined. In those cases where (1) was detected, the amounts found were 
usually on the order of 50-200 ng/g tissue which is below the limit of 
detection in this study. These values are low in both relative and abso- 
lute terms. Since the effective concentration of (1) in in vitro systems 
is on the order of 1.0 yg/g, these levels would be ineffective therapeuti- 
cally. These studies also indicate berberine is not absorbed to a great 
extent from the intestine, again reflecting its highly polar nature. 



125 




126 

The tissue distribution of di hydro be rberine hydrochloride (3) was 
investigated in an analogous manner. The compound (3) is taken up by tis- 
sues to a greater extent than (1) and is handled well by the kidney. The 
concentration of (1) observed in the brain is high. A comparison of the 
time courses of (1) after administration of either (1) or (3), in various 
organs appears in Figures 3-24 to 3-26. These results shown are consistent 
with the greater ability of (3) to penetrate tissues. The liver (Figure 
3-26) is noteworthy because (1) is known to be significantly excreted by 
a biliary mechanism. 

In order to better illustrate the specificity of the (1) Z (2) sys- 
tem, (3) was infused intravenously. The results of these infusions are 
presented in Table 3-18. In these administrations the standard dose of 
55 mg/Kg of (3) was given over a period of either thirty or forty-five 
minutes. If a comparison is made between the concentration of (1) in 
various organs at the end of the infusion and the concentration of (1) 
obtained at thirty or forty- five minutes after a bolus iv injection, the 
specificity inherent in the system can be demonstrated. If the system 
is not specific, one would expect to see an overall decrease in the con- 
centration of (1) after an iv infusion of (3) compared to the bolus. 
The results show that at thirty minutes, there is a higher concentration 
of (1) in the brain compared to the iv bolus and a decrease in the concen- 
tration of (1) in the lungs and kidneys. The liver shows a moderate in- 
crease. At forty-five minutes these trends continue. A comparison of 
the data at thirty and forty- five minutes shows an increase of the con- 
centration of (1) in the brain and a reduction in all other organs. The 
concentration of (JJ_ in th£ brain is higher than in any other tissue ana- 
lyzed at forty- five minutes. 



127 



BERBERINE/GRAM TISSUE 




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131 



Table 3-18. Slow Infusion of Dihydroberberine (3) 



Organ Concentration (yg/g) 

after infusion 

Brain 135.95 ± 13 

Kidney 185.5 ±26 

Lung 71.4 ± 10 

Liver 101.2 ± 23 



30 min 



Concentration (yg/g) A(yg/g) 
30 min after iv bolus 

91.8 ±20 +44 
351 .8 ± 54 -166 
210.2 ± 14 -139 

67.8 ±11 +33 



Organ Concentration (yg/g) 

after infusion 

Brain 162.2 ± 8 

Kidney 121.4 ± 19 

Lung 62.8 ± 6 

Liver 79.4 ± 10 



45 min 



Concentration (yg/g) A 
45 min after iv bolus 

88 + 74 

315 -194 

165 -102 

52 + 27 



132 



These data verify the drug delivery scheme devised. After adminis- 
tration of (3) high concentrations of (1) are obtained specifically in 
the brain, while the concentrations in other organs are reduced. The 
reason that the slow infusion of (3) enhances the specificity is related 
to a number of factors. Dihydroberberine is a lipophilic compound. The 
iv bolus injection presents to the tissues a large concentration of (2) 
and the result of this large burden is an inordinately long transit time 
of (2) in peripheral sites. This obfuscates the kinetics. By slowly 
administering (3), the tissue burden is greatly reduced, and the designed 
improvements in the bidirectional characteristics of the delivery molecule 
can be fully demonstrated. This system in general, and (2) in particular, 
allows specific delivery of quaternary compounds to the brain. This sys- 
tem is designed to reduce systemic levels of an agent and thereby reduce 
any accompanying toxicity. 
Efflux of Berberine from the Brain 

The mechanism by which berberine leaves the brain is not known but 
it is important to the quaternary scheme. According to the original postu- 
lation, large quaternary compounds like (2) should leave the brain slowly, 
presumably by passive processes such as movement in the CSF. Small qua- 
ternary compounds, on the other hand, are substrates for active carriers 
which rapidly remove these compounds from the brain extracellular fluid."*^ 
The next series of experiments was designed to determine the mechanism of 
efflux of (1) from the brain. If the efflux of (2) is mediated by a spe- 
cific carrier, it should be possible to demonstrate the competitive inhi- 
bition of the efflux of (2) by introducing into the system a large con- 
centration of another agent which has affinity for the cationic pump, such 
as 1 -methyl nicotinamide (6) and 1 -benzyl nicotinamide (7). 



133 

The first experiments involved injecting rats with 200 mg/Kg of 
1 -methyl -1 ,4-dihydronicotinamide (21) in aqueous ethanol followed fifteen 
minutes later by an injection of the standard dose of 55 mg/Kg of (3). 
The results of this study are shown in Figure 3-27. The dihydronicotin- 
amide (21) was used as a proform of quaternary compound (6) in an attempt 
to generate high levels of (6) in the brain. The results do not show, how- 
ever, any significant difference in the efflux of berberine (1) between 
pretreated and unpretreated animals. 

In an analogous study 1 -benzyl nicotinamide (7) and its corresponding 
dihydro adduct (22) was employed. Unlike the 1-methyl derivatives, this 
pair of compounds possesses a UV chromophore which greatly simplifies quan- 
titation. A preliminary study was performed to determine the time at which 
the maximum concentration {t^^ax) °^ ^^^^^ administration of 1- 
benzyl-1 ,4-dihydronicotinamide (22) occurred. The results of this experi- 
ment are shown in Figure 3-28. After injection of (7) no detectable levels 
of (7) in the brain could be observed. The reason the t^^^^ is important 
is that, ideally, the lag time between the injection of the antagonist, 
i.e. (7) and (2) is the time required for (7) to reach a maximum concen- 
tration. Unfortunately, however, the toxicity of dihydrobenzyl derivative 
(22) proved to be much higher than that of dihydromethyl derivative (21) 
and the maximum dose which could be given was only 60 mg/Kg and, because 
of this, further studies with this compound were abandoned. 

A number of observations concerning this figure are germane to the 
topic of the efflux of (1) from the brain. In the case of administration 
of this N-benzyldihydropyridine (22), no dihydronicotinamide is present at 
early time points at the dose used in the experiment. This is not the 
case with (2). This is consistent with the greater stability of (31) and 
hence, (2), predicted by the theoretical calculations. The of efflux 



134 




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135 




136 



from the N-benzyl quaternary compound (7) from the brain is 5.8 hours, 
which is twice as fast as the efflux of (1). 

These studies indicate that there was no effect of the quaternary 
compound N-methylnicotinamide (6) on the efflux of (1) from the brain. 
The concentration difference, however, between (6) and (2) was only a 
factor of four at the injection, and this difference is even less at the 
level of the brain. To overcome this quantitative objection, a series of 
intracerebral ventricular (icv) injections was performed. The purpose of 
injecting the compound icv was to allow direct introduction of high con- 
centrations of (1) and (1) with the putative competitive inhibitor, (6), 
into the brain. The injections were made into the lateral ventricles of 
rats with the aid of a stereotaxic instrument and an infusion pump. A 
dose of 50 ug of (1) or 50 yg of (1) and 1000 yg of (6) was administered 
and the compounds were dissolved in DMSO. The volume of the dose was 3-5 
yl . The results of these studies are shown in Figure 3-29. It can be 
seen that (6) has little effect on the efflux of (1) from the brain. The 
ti obtained from the terminal portion of the curves was 3,8 hours for the 

2 

efflux of (1) and 3.1 hours for the efflux of (1) when coinjected with (6). 
The fact that the t^ obtained for (1) in this experiment is much slower 

2 

than that obtained when (1) is administered systemically as (3) is not 
surprising. These icv injections are similar to intrathecal injections 
in that even or complete distribution does not occur. Therefore, (1) is 
basically restricted to the CSF. In the case of systemic administration 
of (3), however, the distribution of (2) is fairly complete and even in 
the brain. The efflux of (1) after administration of (3) is limited by 
the diffusion of (1) from the brain tissue, and from the discussion of the 
BBB, this is a slow and inefficient process. When (1) is administered icv. 



137 



I 




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-(-> 
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138 

the limitations of diffusion do not apply and (1) may simply be lost by 
the bulk flow of CSF. 

To determine the rate of CSF flow in the animal system used in these 
experiments, the rate of efflux of ^H-inulin (29) from the brain was mea- 
sured. This radionuclide was injected icv in DMSO at a dose of 2.3 yCi . 
As shown in Table 3-19, the ti of efflux of (29) is two hours, in good 

z 

agreement with previously reported values.'*^ 

The loss of (1) from the brain appears not to be mediated by an ac- 
tive process. Simple bulk flow of CSF seems to be sufficient to remove 
(1) from the brain. The rate-limiting step in the cerebral elimination 
of (1) does not appear to be the actual efflux process but rather, the 
redistribution of (1) out of brain cells and membranes by this poorly 
mobile species to those areas where elimination can take place. 
Limited Metabolic Studies 

The metabolism of (1) after the administration of (1) or (3) was 
studied in the rat. Urine collected for three days after a dosing of (1) 
or (3) was extracted with chloroform or 3-methyl -1-butanol . The alcohol 
was used because it is reported to extract berberine and similar alkaloids 
efficiently from aqueous sol utions .^^o The results of the HPLC and TLC 
analyses are shown in Tables 3-20 and 3-21, respectively. As one can see, 
there is very little metabolism of (1) and the major component of the 
urine from animals dosed with either (1) or (3) was (1). There were sev- 
eral peaks which could not be attributed to the anesthetic and these were 
found in the urine of all tested animals. The size and retention time 
of these peaks was again similar. 

These data are consistent with the few articles which have been pub- 
lished concerning the metabolism of (1). While this metabolism is reported 



139 



Table 3-19. Efflux of ^H-Inulin from the Brain after Intracerebral 
Ventricular Administration 



Time (min) % Dose/g In % Dose/g 

Wet Tissue Wet Tissue 



15 9.28% 2.23 

30 7.94% 2.07 

45 6.52% 1.87 

60 5.54% 1.71 

120 4.71% 1.55 

Corr. = 0.930 k = 6.23 x lO'^min"! t^^ = 1.9 hrs 



140 



Table 3-20. In Vivo Metabolism of Berberine and Dihydroberberine in the Rat 
(HPLC) 



HPLCa 
Compound 



Retention time of 
Peaks Observed (min) 



Peak Height 
Relative to Berberine 



Berberine 

Chloroform Extract 



1.8^ 

2.0^ 

2.5^ 

2.9 

4.2 

5.6 



0.73 

0.44 

0.39 

0.05 

1.0 

0.01 



Isoamyl Alcohol 
Extract 



Di hydro berberine 
Chloroform Extract 



1.7^ 
2.5^ 
3.6 

* 

4.0 
4.8 

1.8^ 
2.1^^ 
2.6^ 
3.0 

* 

4.2 
5.6 



4.7 
1.4 

0.28 

1.0 

0.09 

0.52 

0.37 

0.35 

0.04 

1.0 

0.01 



141 



Table 3 -20- continued. 



HPLC 



Pi hydro be rberine 

Isoamyl Alcohol 
Extract 



Retention Time of Peak Height 



Compound Peaks Observed (min) Relative to Berberine 



1.9^ 


5.1 


2.5^ 


1.3 


3.4 


0.18 


4.1* 


1.0 


4.8 


0.09 



*Corresponds to berberine standard 

XThese peaks were found in blank animals injected with the anesthetic 
a 

Five microliters of the sample were injected on a yBondapak Ciq reverse 
phase column. The mobile phase was acetonitrile:pH 6.2 phosphate buffer 
60:40 and the flow rate was 2.0 ml /min 



142 



Table 3-21. In Vivo Metabolism of Berberine and Dihydroberberine in 
the Rat (TLC) 

TLC ^ 

Compound Rx of Spots Observed 



Berberine 

Chloroform Extract 0.15* 

O.125X 
0.21^ 
0.34 

Isoamyl Alcohol 0.14* 
Extract 

0.34 

Dihydroberberine 

Chloroform Extract 0.21* 

0.35 

Isoamyl Alcohol 0.21* 
Extract 

0.26^ 

0.29 

0.60 

*Corresponds to berberine standard 

^These spots were found in blank animals injected with the anesthetic 

^Five microliters of the sample were spotted on alumina plates and eluted 
with cyclohexane: chloroform: acetic acid 45:45:10 



143 

be to minimal in vivo , two minor metabolites have been identified. Furuya 
described a urinary metabolite which apparently contained a carboxylic acid 
moiety. Another study reported that very small amounts of tetrahydro- 
berberine were present in the urine. Identification of the metabolites 
found in the present study was not attempted. 

The importance of these studies to the proposed drug delivery scheme 
is related to the requirement of the scheme that the principal and, ideally, 
only metabolism of (2) is to (1). This was shown to be the case. In ad- 
dition, no metabolite was present in the urine extracts of animals dosed 
with (3) which was not present in the urine extracts of animals dosed 
with (1). 

Toxicity and Anticancer Activity of Dihydroberberine 

The toxicity of (1) and (3) was determined in mice and the results 
are shown in Figure 3-30 and Table 3-22. The data were analyzed by the 
method of Probits as well as by fitting the data to a sigmoid dose- response 
curve. The lethal dose for 50% mortality (LD50) for (1) was found to be 
37.0 mg/Kg, and that of (3), 58.2 mg/Kg. The injections were made in- 
traperitoneal ly. The value obtained for (1) was in good agreement with 
other values reported in the 1 iterature. '2i2 j^e toxicity of (1) is 
60% higher than that of (3), substantiating a prediction made by the 
theoretical calculations. The anticancer activity of (1) and (3) is 
presented in Table 3-23. First, the ability of (1) or (3) to inhibit 
the growth of KB cells in vitro was investigated. The ID50 calculated 
for (3) was 2.2 yg/ml while that calculated for (1) was 0.95 ug/ml . 
This is again constant with the higher toxicity of (1) relative to (3). 

The next series of experiments involved inoculating mice with P388 
lymphocytic leukemia cells. If this is done ip, (1) and (3) are equi potent 



144 



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145 



Table 3-22. Probit Analysis of the LD50 Study 



Dose In [Dose] % Dead Probit 



Berberi ne 



Dihydroberberine 
Hydrochlori de 



10 


2.303 





17 


2.833 





24 


3.178 


10 


31 


3.434 


20 


38 


3.637 


50 


45 


3.807 


70 


59 


4.078 


90 


80 


4.382 


100 


25 


3.219 





33.3 


3.506 





41.6 


3.728 


10 


50 


3.912 


30 


58.3 


4.066 


40 


66.5 


4.199 


60 


75 


4.317 


80 


83.3 


4.422 


90 


100 


4.605 


100 



3.718 
4.150 
5.000 
5.524 
6.282 
8.719 



3.718 
4.476 
4.747 
5.253 
5.842 
6.282 
8.719 



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147 

in increasing the life span (ILS) of the animals compared to a control 
group. However, when the leukemia is inoculated intracerebrally , (3) is 
significantly more effective in increasing life span than is (1). The 
% ILS actually falls when (1) is administered. This is indicative of 
the toxic peripheral effects of (1). These preliminary anticancer studies 
again support the original hypothesis. The dihydropyridine is capable of 
passing the BBB and oxidizing to (1) where it may exert its cytotoxic 
acti vity. 

Conclusions 

This dissertation has presented a broadly applicable drug delivery 
scheme which is specific for the brain. This delivery method is based on 
a dihydropyridine-pyridinium salt redox system and on the multi faceted 
nature of the BBB. There are two major aspects of this delivery scheme. 
In the first, which is a chemical delivery system, a pyridinium carrier 
is attached to a drug molecule. The second, and the one with which this 
dissertation has dealt, is a prodrug system in which the carrier moiety 
is an integral component of the molecule. This system is simpler than the 
first but since the molecule to be delivered must contain a pyridinium par- 
tial structure, it is less general. In both cases the basis of the brain 
specific delivery is related to the greater lipophilicity of dihydropyri- 
dines, the ease of their oxidation and subsequent elimination peripherally 
and the difficulty with which large pyridinium compounds leave the CNS. 

In order to substantiate the proposed method, it was applied to a 
salient example, berberine (1). This anticancer alkaloid contains a pyri- 
dinium moiety which is reducible and whose product of reduction is stable. 
The physical and chemical properties of dihydroberberine (2) were examined. 
Its rate of oxidation was found to be rapid in a number of media but not 
as rapid as the rate of oxidation of simple dihydropyridines . Dihydroberberi 



148 

was shown to be more lipophilic than berberine (1) and also better able to 
penetrate biological membranes. In an attempt to delve into the basic 
chemistry of these relatively unstable compounds, a model system was de- 
veloped for them and this was examined by a MINDO/3 approach. The results 
obtained from this study were consistent with experimental data and were 
extendable to the berberine (1) ^ dihydroberberine (2) pair. In addition, 
several predictions were made concerning the biological activity of (2) 
relative to (1) and these were found to be valid. 

Dihydroberberine (2) or its hydrochloride salt (3) was injected iv 
into rats and when the brains were analyzed, high levels of (1) were found. 
No berberine (1) was found in the brain after systemic administration of 
(1). The rate at which (1) left the brain after its delivery by (3) was 
slow and the of the efflux was eleven hours. If dihydroberberine hy- 
drochloride (3) is slowly infused iv, the concentration of (1) rises in 
the brain with time but falls in all other organs tested. At forty-five 
minutes the concentration of (1) is highest in the brain. The efflux of 
berberine (1) from the CNS appears to be mediated by a passive process, 
perhaps the bulk flow of CSF. 

Dihydroberberine hydrochloride (3) was shown to be less toxic than 
(1) in vivo in accordance with predictions made by the theoretical studies. 
Additionally, (3) was shown to be less effective than (1) in inhibiting 
the growth of KB cells in vitro . While the two compounds are equipotent 
in increasing the life span of mice injected ip with a suspension of P388 
lymphocytic leukemia cell, (3) is more potent in increasing the life span 
of mice who were inoculated intracerebral ly with the P388 cell line. 

These data verify the proposed drug delivery scheme. By concentrat- 
ing a pharmacologically active agent at its site of action and by reducing 
its concentration in other locations, the therapeutic index of the delivered 



149 

compound is greatly enhanced. This was demonstrated when the delivery 
scheme was applied to the berberine (1 )Jdihydroberberine (2) example. 

This method is potentially extendable to any drug which contains a 
pyridinium moiety. A number of anticancer agents such as nitidine, cora 
lyne, and fagaronine fit these criteria. Phenothiazines and 3-blockers, 
among others, could also be modified in this way to attain specific deli 
ery to the brain. 



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BIOGRAPHICAL SKETCH 
Marcus Eli Brewster III was bom in Jacksonville, Florida, on October 
14, 1957, the 891st anniversary of the Battle of Hastings. He graduated 
from S. Wolf son High School in 1975. He then enrolled at Mercer Univer- 
sity in Macon, Georgia, where he earned his B.S. cum laude in 1978 with a 
major in biology and a minor in chemistry. At Mercer he was a member of 
Lambda Chi Alpha social fraternity. Beta Beta Beta biological honor so- 
ciety and Gamma Sigma Epsilon chemical fraternity. After a summer posi- 
tion as a historian of the War between the States, he entered graduate 
school at the University of Florida, College of Pharmacy. Four years 
later he was granted a Ph.D. He is a member of American Chemical Society, 
American Pharmaceutical Association, American Association for Advancement 
of Science, and Society for Applied Spectroscopy. 



160 



I certify that I have read this study and that in my opinion 
it conforms to acceptable standards of scholarly presentation and 
is fully adequate, in scope and quality, as a dissertation for the 
degree of Doctor of Philosophy. 

Nicholas S. Bodor, Chairman 
Professor of Medicinal Chemistry 



I certify that I have read this study and that in my opinion 
it conforms to acceptable standards of scholarly presentation and 
is fully adequate, in scope and quality, as a dissertation for the 
degree of Doctor of Philosophy. 




Kenneth B. 
Assistant Professor of 
Medicinal Chemistry 



I certify that I have read thi? study and that in my opinion 
it conforms to acceptable standards of scholarly presentation and 
is fully adequate, in scope and quality, as a dissertation for the 
degree of Doctor of Philosophy. 




Margaret 0. James 
Assistant Professor of 
Medicinal Chemistry 



I certify that I have read this study and that in my opinion 
it conforms to acceptable standards of scholarly presentation and 
is fully adequate, in scope and quality, as a dissertation for the 
degree of Doctor of Philosophy. 



I. Srmpkin/^ 



^mes W. 

>sistant Professor of 
'Pharmacy 

I certify that I have read this study and that in my opinion 
it conforms to acceptable standards of scholarly presentation and 
is fully adequate, in scope and quality, as a dissertation for the 
degree of Doctor of Philosophy. ^ 



Merle A. Battiste 
Professor of Chemistry 



This dissertation was submitted to the Graduate Faculty of the College 
of Pharmacy and to the Graduate Council, and was accepted as partial 
fulfillment of the requirements for the degree of .Doctor of Philosophy. 



August 1982 




Dean, 



ge' of Pharmacy 



Dean for Graduate Studies 
and Research